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Abstract:

The present invention relates to antisense oligonucleotides that modulate
the expression of and/or function of Insulin Receptor Substrate 2 (IRS2)
polynucleotides, in particular, by targeting natural antisense
polynucleotides of Insulin Receptor Substrate 2 (IRS2) polynucleotides
and Transcription factor E3 (TFE3). The invention also relates to the
identification of these antisense oligonucleotides and their use in
treating diseases and disorders associated with the expression of IRS2.

Claims:

1. A method of modulating a function of and/or the expression of a
Insulin Receptor Substrate 2 (IRS2) polynucleotide in patient cells or
tissues in vivo or in vitro comprising: contacting said cells or tissues
with at least one antisense oligonucleotide 5 to 30 nucleotides in length
wherein said at least one oligonucleotide has at least 50% sequence
identity to a reverse complement of a polynucleotide comprising 5 to 30
nucleotides within nucleotides: 1 to 497 of SEQ ID NO: 2 or nucleotides 1
to 633 of SEQ ID NO: 3; thereby modulating a function of and/or the
expression of the Insulin Receptor Substrate 2 (IRS2) polynucleotide in
patient cells or tissues in vivo or in vitro.

2. A method of modulating a function of and/or the expression of a
Insulin Receptor Substrate 2 (IRS2) polynucleotide in patient cells or
tissues in vivo or in vitro comprising: contacting said cells or tissues
with at least one antisense oligonucleotide 5 to 30 nucleotides in length
wherein said at least one oligonucleotide has at least 50% sequence
identity to a reverse complement of a natural antisense of a
Transcription factor E3 (TFE3) polynucleotide or Insulin Receptor
Substrate 2 (IRS2); thereby modulating a function of and/or the
expression of the Insulin Receptor Substrate 2 (IRS2) polynucleotide in
patient cells or tissues vivo or in vitro.

3. A method of modulating a function of and/or the expression of a
Insulin Receptor Substrate 2 (IRS2) polynucleotide patient cells or
tissues in vivo or in vitro comprising: contacting said cells or tissues
with at least one antisense oligonucleotide 5 to 30 nucleotides in length
wherein said oligonucleotide has at least 50% sequence identity to an
antisense oligonucleotide to the Transcription factor E3 (TFE3)
polynucleotide or Insulin Receptor Substrate 2 (IRS2); thereby modulating
a function of and/or the expression of the Insulin Receptor Substrate 2
(IRS2) polynucleotide in patient cells or tissues in vivo or in vitro.

4. A method of modulating a function of and/or the expression of a
Receptor Substrate 2 (IRS2) polynucleotide in patient cells or tissues in
vivo or in vitro comprising: contacting said cells or tissues with at
least one antisense oligonucleotide that targets a region of a natural
antisense oligonucleotide of the Transcription factor E3 (TFE3) or
insulin Receptor Substrate 2 (IRS2) polynucleotide; thereby modulating a
function of and/or the expression of the Insulin Receptor Substrate 2
(IRS2) polynucleotide in patient cells or tissues in vivo or in vitro.

5. The method of claim 4, wherein a function of and/or the expression of
the Insulin Receptor Substrate 2 (IRS2) is increased in vivo or in vitro
with respect to a control.

9. The method of claim 4, wherein the at least one antisense
oligonucleotide comprises one or more modifications selected from: at
least one modified sugar moiety, at least one modified internucleoside
linkage, at least one modified nucleotide, and combinations thereof.

10. The method of claim 9, wherein the one or more modifications comprise
at least one modified sugar moiety selected from: a 2'-O-methoxyethyl
modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl
modified sugar moiety, a bicyclic sugar moiety, and combinations thereof.

12. The method of claim 9, wherein the one or more modifications comprise
at least one modified nucleotide selected from: a peptide nucleic acid
(PNA), a locked nucleic acid (LNA), an arabino-nucleic acid (FANA), an
analogue, a derivative, and combinations thereof.

13. The method of claim 1, wherein the at least one oligonucleotide
comprises at least one oligonucleotide sequences set forth as SEQ ID NOS:
4 to 9.

14. A method of modulating a function of and/or the expression of a
Insulin Receptor Substrate 2 (IRS2) gene in mammalian cells or tissues in
vivo or in vitro comprising: contacting said cells or tissues with at
least one short interfering RNA (siRNA) oligonucleotide 5 to 30
nucleotides in length, said at least one siRNA oligonucleotide being
specific for an antisense polynucleotide of a Transcription factor E3
(TFE3) or Insulin Receptor Substrate 2 (IRS2) polynucleotide, wherein
said at least one siRNA oligonucleotide has at least 50% sequence
identity to a complementary sequence of at least about five consecutive
nucleic acids of the antisense and/or sense nucleic acid molecule of the
Transcription factor E3 (TFE3) or Insulin Receptor Substrate 2 (IRS2)
polynucleotide; and, modulating a function of and/or the expression of or
Insulin Receptor Substrate 2 (IRS2) in mammalian cells or tissues in vivo
or in vitro.

15. The method of claim 14, wherein said oligonucleotide has at least 80%
sequence identity to a sequence of at least about five consecutive
nucleic acids that is complementary to the antisense and/or sense nucleic
acid molecule of the Transcription factor E3(TFE3) or Insulin Receptor
Substrate 2 (IRS2) polynucleotide.

16. A method of modulating a function of and/or the expression of Insulin
Receptor Substrate 2 (IRS2) in mammalian cells or tissues in vivo or in
vitro comprising: contacting said cells or tissues with at least one
antisense oligonucleotide of about 5 to 30 nucleotides in length specific
for noncoding and/or coding sequences of a sense and/or natural antisense
strand of a Transcription factor E3 (TFE3) or Insulin Receptor Substrate
2 (IRS2) polynucleotide wherein said at least one antisense
oligonucleotide has at least 50% sequence identity to at least one
nucleic acid sequence set forth as SEQ ID NOS: 1 to 3; and, modulating
the function and/or expression of the Insulin Receptor Substrate 2 (IRS2)
in mammalian cells or tissues in vivo or in vitro.

17. A synthetic, modified oligonucleotide comprising at least one
modification wherein the at least one modification is selected from: at
least one modified sugar moiety; at least one modified internucleotide
linkage; at least one modified nucleotide, and combinations thereof;
wherein said oligonucleotide is an antisense compound which hybridizes to
and modulates the function and/or expression of a Insulin Receptor
Substrate 2 (IRS2) gene in vivo or in vitro as compared to a normal
control.

26. The oligonucleotide of claim 17, wherein the oligonucleotide is of at
least about 5 to 30 nucleotides in length and hybridizes to an antisense
and/or sense strand of a Transcription factor E3 (TFE3) or Insulin
Receptor Substrate 2 (IRS2) wherein said oligonucleotide has at least
about 20% sequence identity to a complementary sequence of at least about
five consecutive nucleic acids of the antisense and/or sense coding
and/or noncoding nucleic acid sequences of the Transcription factor E3
(TFE3) or Insulin Receptor Substrate 2 (IRS2) polynucleotide.

27. The oligonucleotide of claim 17, wherein the oligonucleotide has at
least about 80% sequence identity to a complementary sequence of at least
about five consecutive nucleic acids of the antisense and/or sense coding
and/or noncoding nucleic acid sequence of the Transcription factor E3
(TFE3) or Insulin Receptor Substrate 2 (IRS2) polynucleotide.

28. The oligonucleotide of claim 17, wherein said oligonucleotide
hybridizes to and modulates expression and/or function of at least one
insulin Receptor Substrate 2 (IRS2) polynucleotide in vivo or in vitro,
as compared to a normal control.

29. The oligonucleotide of claim 17, wherein the oligonucleotide
comprises the sequences set forth as SEQ ID NOS: 4 to 9.

35. A method of preventing or treating a disease associated with at least
one Insulin Receptor Substrate 2 (IRS2) polynucleotide and/or at least
one encoded product thereof, comprising: administering to a patient a
therapeutically effective dose of at least one antisense oligonucleotide
that binds to a natural antisense sequence of said at least one
Transcription factor E3 (TFE3) or Insulin Receptor Substrate 2 (IRS2)
polynucleotide and modulates expression of said at least one Insulin
Receptor Substrate 2 (IRS2) polynucleotide; thereby preventing or
treating the disease associated with the at least one Insulin Receptor
Substrate 2 (IRS2) polynucleotide and/or at least one encoded product
thereof.

37. A method of identifying and selecting at least one oligonucleotide
for in vivo administration comprising: selecting a target polynucleotide
associated with a disease state; identifying at least one oligonucleotide
comprising at least five consecutive nucleotides which are complementary
to the selected target polynucleotide or to a polynucleotide that is
antisense to the selected target polynucleotide; measuring the thermal
melting point of a hybrid of an antisense oligonucleotide and the target
polynucleotide or the polynucleotide that is antisense to the selected
target polynucleotide under stringent hybridization conditions; and
selecting at least one oligonucleotide for in vivo administration based
on the information obtained.

Description:

[0001] The present application claims the priority of U.S. provisional
patent application No. 61/291,419 filed Dec. 31, 2009 which is
incorporated herein by reference in its entirety.

[0003] DNA-RNA and RNA-RNA hybridization are important to many aspects of
nucleic acid function including DNA replication, transcription, and
translation. Hybridization is also central to a variety of technologies
that either detect a particular nucleic acid or alter its expression.
Antisense nucleotides, for example, disrupt gene expression by
hybridizing to target RNA, thereby interfering with RNA splicing,
transcription, translation, and replication. Antisense DNA has the added
feature that DNA-RNA hybrids serve as a substrate for digestion by
ribonuclease H, an activity that is present in most cell types. Antisense
molecules can be delivered into cells, as is the case for
oligodeoxynucleotides (ODNs), or they can be expressed from endogenous
genes as RNA molecules. The FDA recently approved an antisense drug,
VITRAVENE® (for treatment of cytomegalovirus retinitis), reflecting
that antisense has therapeutic utility.

SUMMARY

[0004] This Summary is provided to present a summary of the invention to
briefly indicate the nature and substance of the invention. It is
submitted with the understanding that it will not be used to interpret or
limit the scope or meaning of the claims.

[0005] In one embodiment, the invention provides methods for inhibiting
the action of a natural antisense transcript by using antisense
oligonucleotide(s) targeted to any region of the natural antisense
transcript resulting in up-regulation of the corresponding sense gene. It
is also contemplated herein that inhibition of the natural antisense
transcript can be achieved by siRNA, ribozymes and small molecules, which
are considered to be within the scope of the present invention.

[0006] One embodiment provides a method of modulating function and/or
expression of an IRS2 polynucleotide in patient cells or tissues in vivo
or in vitro comprising contacting said cells or tissues with an antisense
oligonucleotide 5 to 30 nucleotides in length wherein said
oligonucleotide has at least 50% sequence identity to a reverse
complement of a polynucleotide comprising 5 to 30 consecutive nucleotides
within nucleotides 1 to 497 of SEQ ID NO: 2 or nucleotides 1 to 633 of
SEQ ID NO: 3 thereby modulating function and/or expression of the IRS2
polynucleotide in patient cells or tissues in vivo or in vitro.

[0007] In an embodiment, an oligonucleotide targets a natural antisense
sequence of IRS2 or TFE3 polynucleotides, for example, nucleotides set
forth in SEQ ID NOS: 2 and 3, and any variants, alleles, homologs,
mutants, derivatives, fragments and complementary sequences thereto.
Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 4 to
9.

[0008] Another embodiment provides a method of modulating function and/or
expression of an IRS2 polynucleotide in patient cells or tissues in vivo
or in vitro comprising contacting said cells or tissues with an antisense
oligonucleotide 5 to 30 nucleotides in length wherein said
oligonucleotide has at least 50% sequence identity to a reverse
complement of the an antisense of the IRS2 or TFE3 polynucleotide;
thereby modulating function and/or expression of the IRS2 polynucleotide
in patient cells or tissues in vivo or in vitro.

[0009] Another embodiment provides a method of modulating function and/or
expression of an IRS2 polynucleotide in patient cells or tissues in vivo
or in vitro comprising contacting said cells or tissues with an antisense
oligonucleotide 5 to 30 nucleotides in length wherein said
oligonucleotide has at least 50% sequence identity to an antisense
oligonucleotide to an IRS2 or TFE3 antisense polynucleotide; thereby
modulating function and/or expression of the IRS2 polynucleotide in
patient cells or tissues in vivo or in vitro.

[0010] In an embodiment, a composition comprises one or more antisense
oligonucleotides which bind to sense and/or antisense IRS2 or TFE3
polynucleotides.

[0011] In an embodiment, the oligonucleotides comprise one or more
modified or substituted nucleotides.

[0012] In an embodiment, the oligonucleotides comprise one or more
modified bonds.

[0014] In an embodiment, the oligonucleotides are administered to a
patient subcutaneously, intramuscularly, intravenously or
intraperitoneally.

[0015] In an embodiment, the oligonucleotides are administered in a
pharmaceutical composition. A treatment regimen comprises administering
the antisense compounds at least once to patient; however, this treatment
can be modified to include multiple doses over a period of time. The
treatment can be combined with one or more other types of therapies.

[0016] In an embodiment, the oligonucleotides are encapsulated in a
liposome or attached to a carrier molecule (e.g. cholesterol, TAT
peptide).

[0019] FIG. 2 is a graph of real time PCR results showing the fold
change+standard deviation in IRS2 mRNA after treatment of Vero76 cells
with phosphorothioate oligonucleotides introduced using Lipofectamine
2000, as compared to control. Bars denoted as CUR-0690, CUR-0691, and
CUR-0692 correspond to SEQ ID NOS 6, 7, and 8.

[0020] FIG. 3 is a graph of real time PCR results showing the fold
change+standard deviation in IRS2 mRNA after treatment of MCF7 cells with
phosphorothioate oligonucleotides introduced using Lipofectamine 2000, as
compared to control. Bars denoted as CUR-0690, CUR-0691, CUR-0692, and
CUR-0693 correspond to SEQ ID NOS 6, 7, 8 and 9.

[0021] FIG. 4 is a graph of real time PCR results showing the fold
change+standard deviation in TFE3 mRNA after treatment of HepG2 cells
with phosphorothioate oligonucleotides introduced using Lipofectamine
2000, as compared to control. Bars denoted as CUR-0603 and CUR-0605
correspond to SEQ ID NOS 4 and 5 respectively.

[0023] Several aspects of the invention are described below with reference
to example applications for illustration. It should be understood that
numerous specific details, relationships, and methods are set forth to
provide a full understanding of the invention. One having ordinary skill
in the relevant art, however, will readily recognize that the invention
can be practiced without one or more of the specific details or with
other methods. The present invention is not limited by the ordering of
acts or events, as some acts may occur in different orders and/or
concurrently with other acts or events. Furthermore, not all illustrated
acts or events are required to implement a methodology in accordance with
the present invention.

[0024] All genes, gene names, and gene products disclosed herein are
intended to correspond homologs from any species for which the
compositions and methods disclosed herein are applicable. Thus, the terms
include, but are not limited to genes and gene products from humans and
mice. It is understood that when a gene or gene product from a particular
species is disclosed, this disclosure is intended to be exemplary only,
and is not to be interpreted as a limitation unless the context in which
it appears clearly indicates. Thus, for example, for the genes disclosed
herein, which in some embodiments relate to mammalian nucleic acid and
amino acid sequences are intended to encompass homologous and/or
orthologous genes and gene products from other animals including, but not
limited to other mammals, fish, amphibians, reptiles, and birds. In an
embodiment, the genes or nucleic acid sequences are human.

Definitions

[0025] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of the
invention. As used herein, the singular forms "a", "an" and "the" are
intended to include the plural forms as well, unless the context clearly
indicates otherwise. Furthermore, to the extent that the terms
"including", "includes", "having", "has", "with", or variants thereof are
used in either the detailed description and/or the claims, such terms are
intended to be inclusive in a manner similar to the term "comprising."

[0026] The term "about" or "approximately" means within an acceptable
error range for the particular value as determined by one of ordinary
skill in the art, which will depend in part on how the value is measured
or determined, i.e., the limitations of the measurement system. For
example, "about" can mean within 1 or more than 1 standard deviation, per
the practice in the art. Alternatively, "about" can mean a range of up to
20%, preferably up to 10%, more preferably up to 5%, and more preferably
still up to 1% of a given value. Alternatively, particularly with respect
to biological systems or processes, the term can mean within an order of
magnitude, preferably within 5-fold, and more preferably within 2-fold,
of a value. Where particular values are described in the application and
claims, unless otherwise stated the term "about" meaning within an
acceptable error range for the particular value should be assumed.

[0027] As used herein, the term "mRNA" means the presently known mRNA
transcript(s) of a targeted gene, and any further transcripts which may
be elucidated.

[0028] By "antisense oligonucleotides" or "antisense compound" is meant an
RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA).
For example, if it is an RNA oligonucleotide it binds to another RNA
target by means of RNA-RNA interactions and alters the activity of the
target RNA. An antisense oligonucleotide can upregulate or downregulate
expression and/or function of a particular polynucleotide. The definition
is meant to include any foreign RNA or DNA molecule which is useful from
a therapeutic, diagnostic, or other viewpoint. Such molecules include,
for example, antisense RNA or DNA molecules, interference RNA (RNAi),
micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing
RNA and agonist and antagonist RNA, antisense oligomeric compounds,
antisense oligonucleotides, external guide sequence (EGS)
oligonucleotides, alternate splicers, primers, probes, and other
oligomeric compounds that hybridize to at least a portion of the target
nucleic acid. As such, these compounds may be introduced in the form of
single-stranded, double-stranded, partially single-stranded, or circular
oligomeric compounds.

[0029] In the context of this invention, the term "oligonucleotide" refers
to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic
acid (DNA) or mimetics thereof. The term "oligonucleotide", also includes
linear or circular oligomers of natural and/or modified monomers or
linkages, including deoxyribonucleosides, ribonucleosides, substituted
and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked
nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.
Oligonucleotides are capable of specifically binding to a target
polynucleotide by way of a regular pattern of monomer-to-monomer
interactions, such as Watson-Crick type of base pairing, Hoogsteen or
reverse Hoogsteen types of base pairing, or the like.

[0030] The oligonucleotide may be "chimeric", that is, composed of
different regions. In the context of this invention "chimeric" compounds
are oligonucleotides, which contain two or more chemical regions, for
example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical
region is made up of at least one monomer unit, i.e., a nucleotide in the
case of an oligonucleotides compound. These oligonucleotides typically
comprise at least one region wherein the oligonucleotide is modified in
order to exhibit one or more desired properties. The desired properties
of the oligonucleotide include, but are not limited, for example, to
increased resistance to nuclease degradation, increased cellular uptake,
and/or increased binding affinity for the target nucleic acid. Different
regions of the oligonucleotide may therefore have different properties.
The chimeric oligonucleotides of the present invention can be formed as
mixed structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide analogs as
described above.

[0031] The oligonucleotide can be composed of regions that can be linked
in "register", that is, when the monomers are linked consecutively, as in
native DNA, or linked via spacers. The spacers are intended to constitute
a covalent "bridge" between the regions and have in preferred cases a
length not exceeding about 100 carbon atoms. The spacers may carry
different functionalities, for example, having positive or negative
charge, carry special nucleic acid binding properties (intercalators,
groove binders, toxins, fluorophors etc.), being lipophilic, inducing
special secondary structures like, for example, alanine containing
peptides that induce alpha-helices.

[0032] As used herein "IRS2" and "Insulin Receptor Substrate 2" are
inclusive of all family members, mutants, alleles, fragments, species,
coding and noncoding sequences, sense and antisense polynucleotide
strands, etc.

[0033] As used herein "TFE3" and "Transcription factor E3" are inclusive
of all family members, mutants, alleles, fragments, species, coding and
noncoding sequences, sense and antisense polynucleotide strands, etc.

[0034] As used herein, the words Insulin Receptor Substrate 2, Insulin
Receptor Substrate-2, IRS-2 and IRS2, are considered the same in the
literature and are used interchangeably in the present application.

[0035] As used herein, the words transcription factor E3, TFE3, TFE-3,
RCCP2 and TFEA, are considered the same in the literature and are used
interchangeably in the present application.

[0036] As used herein, the term "oligonucleotide specific for" or
"oligonucleotide which targets" refers to an oligonucleotide having a
sequence (i) capable of forming a stable complex with a portion of the
targeted gene, or (ii) capable of forming a stable duplex with a portion
of a mRNA transcript of the targeted gene. Stability of the complexes and
duplexes can be determined by theoretical calculations and/or in vitro
assays. Exemplary assays for determining stability of hybridization
complexes and duplexes are described in the Examples below.

[0037] As used herein, the term "target nucleic acid" encompasses DNA, RNA
(comprising premRNA and mRNA) transcribed from such DNA, and also cDNA
derived from such RNA, coding, noncoding sequences, sense or antisense
polynucleotides. The specific hybridization of an oligomeric compound
with its target nucleic acid interferes with the normal function of the
nucleic acid. This modulation of function of a target nucleic acid by
compounds, which specifically hybridize to it, is generally referred to
as "antisense". The functions of DNA to be interfered include, for
example, replication and transcription. The functions of RNA to be
interfered, include all vital functions such as, for example,
translocation of the RNA to the site of protein translation, translation
of protein from the RNA, splicing of the RNA to yield one or more mRNA
species, and catalytic activity which may be engaged in or facilitated by
the RNA. The overall effect of such interference with target nucleic acid
function is modulation of the expression of an encoded product or
oligonucleotides.

[0038] RNA interference "RNAi" is mediated by double stranded RNA (dsRNA)
molecules that have sequence-specific homology to their "target" nucleic
acid sequences. In certain embodiments of the present invention, the
mediators are 5-25 nucleotide "small interfering" RNA duplexes (siRNAs).
The siRNAs are derived from the processing of dsRNA by an RNase enzyme
known as Dicer. siRNA duplex products are recruited into a multi-protein
siRNA complex termed RISC (RNA Induced Silencing Complex). Without
wishing to be bound by any particular theory, a RISC is then believed to
be guided to a target nucleic acid (suitably mRNA), where the siRNA
duplex interacts in a sequence-specific way to mediate cleavage in a
catalytic fashion. Small interfering RNAs that can be used in accordance
with the present invention can be synthesized and used according to
procedures that are well known in the art and that will be familiar to
the ordinarily skilled artisan. Small interfering RNAs for use in the
methods of the present invention suitably comprise between about 1 to
about 50 nucleotides (nt). In examples of non limiting embodiments,
siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about
10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.

[0039] Selection of appropriate oligonucleotides is facilitated by using
computer programs that automatically align nucleic acid sequences and
indicate regions of identity or homology. Such programs are used to
compare nucleic acid sequences obtained, for example, by searching
databases such as GenBank or by sequencing PCR products. Comparison of
nucleic acid sequences from a range of species allows the selection of
nucleic acid sequences that display an appropriate degree of identity
between species. In the case of genes that have not been sequenced,
Southern blots are performed to allow a determination of the degree of
identity between genes in target species and other species. By performing
Southern blots at varying degrees of stringency, as is well known in the
art, it is possible to obtain an approximate measure of identity. These
procedures allow the selection of oligonucleotides that exhibit a high
degree of complementarity to target nucleic acid sequences in a subject
to be controlled and a lower degree of complementarity to corresponding
nucleic acid sequences in other species. One skilled in the art will
realize that there is considerable latitude in selecting appropriate
regions of genes for use in the present invention.

[0040] By "enzymatic RNA" is meant an RNA molecule with enzymatic activity
(Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic
acids (ribozymes) act by first binding to a target RNA. Such binding
occurs through the target binding portion of an enzymatic nucleic acid
which is held in close proximity to an enzymatic portion of the molecule
that acts to cleave the target RNA. Thus, the enzymatic nucleic acid
first recognizes and then binds a target RNA through base pairing, and
once bound to the correct site, acts enzymatically to cut the target RNA.

[0041] By "decoy RNA" is meant an RNA molecule that mimics the natural
binding domain for a ligand. The decoy RNA therefore competes with
natural binding target for the binding of a specific ligand. For example,
it has been shown that over-expression of HIV trans-activation response
(TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein,
thereby preventing it from binding to TAR sequences encoded in the HIV
RNA. This is meant to be a specific example. Those in the art will
recognize that this is but one example, and other embodiments can be
readily generated using techniques generally known in the art.

[0042] As used herein, the term "monomers" typically indicates monomers
linked by phosphodiester bonds or analogs thereof to form
oligonucleotides ranging in size from a few monomeric units, e.g., front
about 3-4, to about several hundreds of monomeric units. Analogs of
phosphodiester linkages include: phosphorothioate, phosphorodithioate,
methylphosphomates, phosphoroselenoate, phosphoramidate, and the like, as
more fully described below.

[0043] The term "nucleotide" covers naturally occurring nucleotides as
well as nonnaturally occurring nucleotides. It should be clear to the
person skilled in the art that various nucleotides which previously have
been considered "non-naturally occurring" have subsequently been found in
nature. Thus, "nucleotides" includes not only the known purine and
pyrimidine heterocycles-containing molecules, but also heterocyclic
analogues and tautomers thereof. Illustrative examples of other types of
nucleotides are molecules containing adenine, guanine, thymine, cytosine,
uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine
7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin,
N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,
5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,
pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,
isoguanin, inosine and the "non-naturally occurring" nucleotides
described in Benner et al., U.S. Pat. No. 5,432,272. The term
"nucleotide" is intended to cover every and all of these examples as well
as analogues and tautomers thereof. Especially interesting nucleotides
are those containing adenine, guanine, thymine, cytosine, and uracil,
which are considered as the naturally occurring nucleotides in relation
to therapeutic and diagnostic application in humans. Nucleotides include
the natural 2'-deoxy and 2'-hydroxyl sugars, e.g., as described in
Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco,
1992) as well as their analogs.

[0045] As used herein, "hybridization" means the pairing of substantially
complementary strands of oligomeric compounds. One mechanism of pairing
involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases (nucleotides) of the strands of oligomeric compounds.
For example, adenine and thymine are complementary nucleotides which pair
through the formation of hydrogen bonds. Hybridization can occur under
varying circumstances.

[0046] An antisense compound is "specifically hybridizable" when binding
of the compound to the target nucleic acid interferes with the normal
function of the target nucleic acid to cause a modulation of function
and/or activity, and there is a sufficient degree of complementarity to
avoid non-specific binding of the antisense compound to non-target
nucleic acid sequences under conditions in which specific binding is
desired, i.e., under physiological conditions in the case of in vivo
assays or therapeutic treatment, and under conditions in which assays are
performed in the case of in vitro assays.

[0047] As used herein, the phrase "stringent hybridization conditions" or
"stringent conditions" refers to conditions under which a compound of the
invention will hybridize to its target sequence, but to a minimal number
of other sequences. Stringent conditions are sequence-dependent and will
be different in different circumstances and in the context of this
invention, "stringent conditions" under which oligomeric compounds
hybridize to a target sequence are determined by the nature and
composition of the oligomeric compounds and the assays in which they are
being investigated. In general, stringent hybridization conditions
comprise low concentrations (<0.15M) of salts with inorganic cations
such as Na++ or K++ (i.e., low ionic strength), temperature higher than
20° C.-25° C. below the Tm of the oligomeric
compound:target sequence complex, and the presence of denaturants such as
formamide, dimethylformamide, dimethylsulfoxide, or the detergent sodium
dodecyl sulfate (SDS). For example, the hybridization rate decreases 1.1%
for each 1% formamide. An example of a high stringency hybridization
condition is 0.1× sodium chloride-sodium citrate buffer (SSC)/0.1%
(w/v) SDS at 60' C. for 30 minutes.

[0048] "Complementary," as used herein, refers to the capacity for precise
pairing between two nucleotides on one or two oligomeric strands. For
example, if a nucleobase at a certain position of an antisense compound
is capable of hydrogen bonding with a nucleobase at a certain position of
a target nucleic acid, said target nucleic acid being a DNA, RNA, or
oligonucleotide molecule, then the position of hydrogen bonding between
the oligonucleotide and the target nucleic acid is considered to be a
complementary position. The oligomeric compound and the further DNA, RNA,
or oligonucleotide molecule are complementary to each other when a
sufficient number of complementary positions in each molecule are
occupied by nucleotides which can hydrogen bond with each other. Thus,
"specifically hybridizable" and "complementary" are terms which are used
to indicate a sufficient degree of precise pairing or complementarity
over a sufficient number of nucleotides such that stable and specific
binding occurs between the oligomeric compound and a target nucleic acid.

[0049] It is understood in the art that the sequence of an oligomeric
compound need not be 100% complementary to that of its target nucleic
acid to be specifically hybridizable. Moreover, an oligonucleotide may
hybridize over one or more segments such that intervening or adjacent
segments are not involved in the hybridization event (e.g., a loop
structure, mismatch or hairpin structure). The oligomeric compounds of
the present invention comprise at least about 70%, or at least about 75%,
or at least about 80%, or at least about 85%, or at least about 90%, or
at least about 95%, or at least about 99% sequence complementarity to a
target region within the target nucleic acid sequence to which they are
targeted. For example, an antisense compound in which 18 of 20
nucleotides of the antisense compound are complementary to a target
region, and would therefore specifically hybridize, would represent 90
percent complementarity. In this example, the remaining noncomplementary
nucleotides may be clustered or interspersed with complementary
nucleotides and need not be contiguous to each other or to complementary
nucleotides. As such, an antisense compound which is 18 nucleotides in
length having 4 (four) noncomplementary nucleotides which are flanked by
two regions of complete complementarity with the target nucleic acid
would have 77.8% overall complementarity with the target nucleic acid and
would thus fill within the scope of the present invention. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs (basic
local alignment search tools) and PowerBLAST programs known in the art.
Percent homology, sequence identity or complementarity, can be determined
by, for example, the Gap program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research Park,
Madison Wis.), using default settings, which uses the algorithm of Smith
and Waterman (Adv. Appl. Math., (1981) 2, 482-489).

[0050] As used herein, the term "Thermal Melting Point (Tm)" refers to the
temperature, under defined ionic strength, pH, and nucleic acid
concentration, at which 50% of the oligonucleotides complementary to the
target sequence hybridize to the target sequence at equilibrium.
Typically, stringent conditions will be those in which the salt
concentration is at least about 0.01 to 1.0 M Na ion concentration (or
other salts) at pH 7.0 to 8.3 and the temperature is at least about
30° C. for short oligonucleotides (e.g., 10 to 50 nucleotide).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide.

[0051] As used herein, "modulation" means either an increase (stimulation)
or a decrease (inhibition) in the expression of a gene.

[0052] The term "variant", when used in the context of a polynucleotide
sequence, may encompass a polynucleotide sequence related to a wild type
gene. This definition may also include, for example, "allelic," "splice,"
"species," or "polymorphic" variants. A splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of polynucleotides due to alternate splicing of
exons during mRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species variants
are polynucleotide sequences that vary from one species to another. Of
particular utility in the invention are variants of wild type gene
products. Variants may result from at least one mutation in the nucleic
acid sequence and may result in altered mRNAs or in polypeptides whose
structure or function may or may not be altered. Any given natural or
recombinant gene may have none, one, or many allelic forms. Common
mutational changes that give rise to variants are generally ascribed to
natural deletions, additions, or substitutions of nucleotides. Each of
these types of changes may occur alone, or in combination with the
others, one or more times in a given sequence.

[0053] The resulting polypeptides generally will have significant amino
acid identity relative to each other. A polymorphic variant is a
variation in the polynucleotide sequence of a particular gene between
individuals of a given species. Polymorphic variants also may encompass
"single nucleotide polymorphisms" (SNPs,) or single base mutations in
which the polynucleotide sequence varies by one base. The presence of
SNPs may be indicative of, for example, a certain population with a
propensity for a disease state, that is susceptibility versus resistance.

[0054] Derivative poly-nucleotides include nucleic acids subjected to
chemical modification, for example, replacement of hydrogen by an alkyl,
acyl, or amino group. Derivatives, e.g., derivative oligonucleotides, may
comprise non-naturally-occurring portions, such as altered sugar moieties
or inter-sugar linkages. Exemplary among these are phosphorothioate and
other sulfur containing species which are known in the art. Derivative
nucleic acids may also contain labels, including radionucleotides,
enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents,
substrates, cofactors, inhibitors, magnetic particles, and the like.

[0055] A "derivative" polypeptide or peptide is one that is modified, for
example, by glycosylation, pegylation, phosphorylation, sulfation,
reduction/alkylation, acylation, chemical coupling, or mild formalin
treatment. A derivative may also be modified to contain a detectable
label, either directly or indirectly, including, but not limited to, a
radioisotope, fluorescent, and enzyme label.

[0057] "Mammal" covers warm blooded mammals that are typically under
medical care (e.g., humans and domesticated animals). Examples include
feline, canine, equine, bovine, and human, as well as just human.

[0058] "Treating" or "treatment" covers the treatment of a disease-state
in a mammal, and includes: (a) preventing the disease-state from
occurring in a mammal, in particular, when such mammal is predisposed to
the disease-state but has not yet been diagnosed as having it; (b)
inhibiting the disease-state, e.g., arresting it development; and/or (c)
relieving the disease-state, e.g., causing regression of the disease
state until a desired endpoint is reached. Treating also includes the
amelioration of a symptom of a disease (e.g., lessen the pain or
discomfort), wherein such amelioration may or may not be directly
affecting the disease (e.g., cause, transmission, expression, etc.).

[0062] TFE3, a basic helix-loop-helix (bHLH) protein, as a transactivator
of metabolic genes that are regulated through an E-box in their
promoters. Adenovirus-mediated expression of TFE3 in hepatocytes in
culture and in vivo strongly activated expression of IRS-2 and Akt and
enhanced phosphorylation of insulin-signaling kinases such as Akt,
glycogen synthase kinase 3β and p70S6 kinase, TFE3 is a bHLH
transcription factor that strongly activates various insulin signaling
molecules, protecting against the development of insulin resistance and
the metabolic syndrome.

[0063] Regulation of IRS-2 is the primary site where TFE3 in synergy with
Foxo1, and SREBP-1c converge. Taken together, TFE3/Foxo1 andSREBP-1c
reciprocally regulate IRS-2 expression and insulin sensitivity in the
liver.

[0064] Members of the IRS-protein family are tyrosine phosphorylated by
the receptors for insulin and IGF-1, as well as certain cytokines
receptors coupled to Janus kinases. At least four IRS-proteins occur in
mammals. IRS-1 and IRS-2 are widely expressed; IRS-3 is restricted to
adipose tissue, β-cells, and possibly liver; and IRS-4 is expressed
in the thymus, brain, and kidney. IRS-proteins have a conserved amino
terminus composed of adjacent pleckstrin homology and
phosphotyrosine-binding domains that mediate coupling to activated
receptor tyrosine kinases.

[0065] In an embodiment, antisense oligonucleotides are used to prevent or
treat diseases or disorders associated with IRS2 family members.
Exemplary Insulin Receptor Substrate 2 (IRS2) mediated diseases and
disorders which can be treated with cell/tissues regenerated from stem
cells obtained using the antisense compounds comprise: a disease or
disorder associated with abnormal function and/or expression of IRS2
and/or TFE3, a neurological disease or disorder (e.g. Alzheimer's
disease, Parkinson's disease, amyotrophic lateral sclerosis etc.), a
disease or disorder associated with insulin resistance, diabetes, an
insulin resistant non diabetic state (e.g., obesity, impaired glucose
tolerance (IGT), Metabolic Syndrome etc.), a hepatic disease or disorder,
a disease or disorder associated with kidney growth and development, a
disease or disorder associated with skeletal muscle growth and/or
metabolism, a disease or disorder associated with carbohydrate
metabolism, a weight disorder, Polycystic Ovary Syndrome,
atherosclerosis, cancer, a disease or disorder associated with apoptosis,
a disease or disorder associated with aging and senescence.

[0066] In an embodiment, modulation IRS2 by one or more antisense
oligonucleotides is administered to a patient in need thereof, for
athletic enhancement and body building.

[0067] In an embodiment, modulation of IRS2 by one or more antisense
oligonucleotides is administered to a patient in need thereof, to prevent
or treat any disease or disorder related to IRS2 or TFE3 abnormal
expression, function, activity as compared to a normal control.

[0068] In an embodiment, the oligonucleotides are specific for
polynucleotides of IRS2, which includes, without limitation noncoding
regions. The IRS2 targets comprise variants of IRS2 and TFE3; mutants of
IRS2 and TFE3, including SNPs; noncoding sequences of IRS2 and TFE3;
alleles, fragments and the like. Preferably the oligonucleotide is an
antisense RNA molecule.

[0069] In accordance with embodiments of the invention, the target nucleic
acid molecule is not limited to IRS2 or TFE3 polynucleotides alone but
extends to any of the isoforms, receptors, homologs, non-coding regions
and the like of IRS2 and TFE3.

[0070] In an embodiment, an oligonucleotide targets a natural antisense
sequence (natural antisense to the coding and non-coding regions) of IRS2
and TFE3 targets, including, without limitation, variants, alleles,
homologs, mutants, derivatives, fragments and complementary sequences
thereto. Preferably the oligonucleotide is an antisense RNA or DNA
molecule.

[0071] In an embodiment, the oligomeric compounds of the present invention
also include variants in which a different base is present at one or more
of the nucleotide positions in the compound. For example, if the first
nucleotide is an adenine, variants may be produced which contain
thymidine, guanosine, cytidine or other natural or unnatural nucleotides
at this position. This may be done at any of the positions of the
antisense compound. These compounds are then tested using the methods
described herein to determine their ability to inhibit expression of a
target nucleic acid.

[0072] In some embodiments, homology, sequence identity or
complementarity, between the antisense compound and target is from about
50%, to about 60%. In some embodiments, homology, sequence identity or
complementarity, is from about 60%, to about 70%. In some embodiments,
homology, sequence identity or complementarity, is from about 70% to
about 80%. In some embodiments, homology, sequence identity or
complementarity, is from about 80% to about 90%. In some embodiments,
homology, sequence identity or complementarity, is about 90%, about 92%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about
100%.

[0073] An antisense compound is specifically hybridizable when binding of
the compound to the target nucleic acid interferes with the normal
function of the target nucleic acid to cause a loss of activity, and
there is a sufficient degree of complementarity to avoid non-specific
binding of the antisense compound to non-target nucleic acid sequences
under conditions in which specific binding is desired. Such conditions
include, i.e., physiological conditions in the case of in vivo assays or
therapeutic treatment, and conditions in which assays are performed in
the case of in vitro assays.

[0074] An antisense compound, whether DNA, RNA, chimeric, substituted etc,
is specifically hybridizable when binding of the compound to the target
DNA or RNA molecule interferes with the normal function of the target DNA
or RNA to cause a loss of utility, and there is a sufficient degree of
complementarily to avoid non-specific binding of the antisense compound
to non-target sequences under conditions in which specific binding is
desired, i.e., under physiological conditions in the case of in vivo
assays or therapeutic treatment, and in the case of in vitro assays,
under conditions in which the assays are performed.

[0075] In an embodiment, targeting of IRS2 or TFE3 including without
limitation, antisense sequences which are identified and expanded, using
for example, PCR, hybridization etc., one or more of the sequences set
forth as SEQ ID NOS: 2 and 3, and the like, modulate the expression or
function of IRS2. In one embodiment, expression or function is
up-regulated as compared to a control. In an embodiment, expression or
function is down-regulated as compared to a control.

[0076] In an embodiment, oligonucleotides comprise nucleic acid sequences
set forth as SEQ ID NOS: 4 to 9 including antisense sequences which are
identified and expanded, using for example, PCR, hybridization etc. These
oligonucleotides can comprise one or more modified nucleotides, shorter
or longer fragments, modified bonds and the like. Examples of modified
bonds or internucleotide linkages comprise phosphorothioate,
phosphorodithioate or the like. In an embodiment, the nucleotides
comprise a phosphorus derivative. The phosphorus derivative (or modified
phosphate group) which may be attached to the sugar or sugar analog
moiety in the modified oligonucleotides of the present invention may be a
monophosphate, diphosphate, triphosphate, alkylphosphate,
alkanephosphate, phosphorothioate and the like. The preparation of the
above-noted phosphate analogs, and their incorporation into nucleotides,
modified nucleotides and oligonucleotides, per se, is also known and need
not be described here.

[0077] The specificity and sensitivity of antisense is also harnessed by
those of skill in the art for therapeutic uses. Antisense
oligonucleotides have been employed as therapeutic moieties in the
treatment of disease states in animals and man. Antisense
oligonucleotides have been safely and effectively administered to humans
and numerous clinical trials are presently underway. It is thus
established that oligonucleotides can be useful therapeutic modalities
that can be configured to be useful in treatment regimes for treatment of
cells, tissues and animals, especially humans.

[0078] In embodiments of the present invention oligomeric antisense
compounds, particularly oligonucleotides, bind to target nucleic acid
molecules and modulate the expression and/or function of molecules
encoded by a target gene. The functions of DNA to be interfered comprise,
for example, replication and transcription. The functions of RNA to be
interfered comprise all vital functions such as, for example,
translocation of the RNA to the site of protein translation, translation
of protein from the RNA, splicing of the RNA to yield one or more mRNA
species, and catalytic activity which may be engaged in or facilitated by
the RNA. The functions may be up-regulated or depending on the functions
desired.

[0079] The antisense compounds, include, antisense oligomeric compounds,
antisense oligonucleotides, external guide sequence (EGS)
oligonucleotides, alternate splicers, primers, probes, and other
oligomeric compounds that hybridize to at least a portion of the target
nucleic acid. As such, these compounds may be introduced in the form of
single-stranded, double-stranded, partially single-stranded, or circular
oligomeric compounds.

[0080] Targeting an antisense compound to a particular nucleic acid
molecule, in the context of this invention, can be a multistep process.
The process usually begins with the identification of a target nucleic
acid whose function is to be modulated. This target nucleic acid may be,
for example, a cellular gene (or mRNA transcribed from the gene) whose
expression is associated with a particular disorder or disease state, or
a nucleic acid molecule from an infectious agent. In the present
invention, the target nucleic acid encodes IRS2 or TFE3.

[0081] The targeting process usually also includes determination of at
least one target region, segment, or site within the target nucleic acid
for the antisense interaction to occur such that the desired effect,
e.g., modulation of expression, will result. Within the context of the
present invention, the term "region" is defined as a portion of the
target nucleic acid having at least one identifiable structure, function,
or characteristic. Within regions of target nucleic acids are segments.
"Segments" are defined as smaller or sub-portions of regions within a
target nucleic acid. "Sites," as used in the present invention, are
defined as positions within a target nucleic acid.

[0083] In an embodiment, the antisense oligonucleotides bind to one or
more segments of Insulin Receptor Substrate 2 (IRS2) or Transcription
factor E3 (TFE3) polynucleotides and modulate the expression and/or
function of IRS2. The segments comprise at least five consecutive
nucleotides of the IRS2 or TFE3 sense or antisense polynucleotides.

[0084] In an embodiment, the antisense oligonucleotides are specific for
natural antisense sequences of IRS2 or TFE3 wherein binding of the
oligonucleotides to the natural antisense sequences of IRS2 or TFE3
modulate expression and/or function of IRS2.

[0085] In an embodiment, oligonucleotide compounds comprise sequences set
forth as SEQ ID NOS: 4 to 9, antisense sequences which are identified and
expanded, using for example, PCR, hybridization etc These
oligonucleotides can comprise one or more modified nucleotides, shorter
or longer fragments, modified bonds and the like. Examples of modified
bonds or internucleotide linkages comprise phosphorothioate,
phosphorodithioate or the like. In an embodiment, the nucleotides
comprise a phosphorus derivative. The phosphorus derivative (or modified
phosphate group) which may be attached to the sugar or sugar analog
moiety in the modified oligonucleotides of the present invention may be a
monophosphate, diphosphate, triphosphate, alkylphosphate,
alkanephosphate, phosphorothioate and the like. The preparation of the
above-noted phosphate analogs, and their incorporation into nucleotides,
modified nucleotides and oligonucleotides, per se, is also known and need
not be described here.

[0086] Since, as is known in the art, the translation initiation codon is
typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the
corresponding DNA molecule), the translation initiation codon is also
referred to as the "AUG codon," the "start codon" or the "AUG start
codon". A minority of genes has a translation initiation codon having the
RNA sequence 5'-GUG, 5'-UUG or or 5'-CUG; and 5'-AUA, 5'-ACG and 5-CUG
have been shown to function in vivo. Thus, the terms "translation
initiation codon" and "start codon" can encompass many codon sequences,
even though the initiator amino acid in each instance is typically
methionine (in eukaryotes) or formylmethionine (in prokaryotes).
Eukaryotic and prokaryotic genes may have two or more alternative start
codons, any one of which may be preferentially utilized for translation
initiation in a particular cell type or tissue, or under a particular set
of conditions. In the context of the invention, "start codon" and
"translation initiation codon" refer to the codon or codons that are used
in vivo to initiate translation of an mRNA transcribed from a gene
encoding Insulin Insulin Receptor Substrate 2 (IRS2) or Transcription
factor E3 (TFE3), regardless of the sequence(s) of such codons. A
translation termination codon (or "stop codon") of a gene may have one of
three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA
sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).

[0087] The terms "start codon region" and "translation initiation codon
region" refer to a portion of such an mRNA or gene that encompasses from
about 25 to about 50 contiguous nucleotides in either direction (i.e., 5'
or 3') from a translation initiation codon. Similarly, the terms "stop
codon region" and "translation termination codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to about
50 contiguous nucleotides in either direction (i.e., 5' or 3') from a
translation termination codon. Consequently, the "start codon region" (or
"translation initiation codon region") and the "stop codon region" (or
"translation termination codon region") are all regions that may be
targeted effectively with the antisense compounds or the present
invention.

[0088] The open reading frame (ORF) or "coding region," which is known in
the art to refer to the region between the translation initiation codon
and the translation termination codon, is also a region which may be
targeted effectively. Within the context of the present invention, a
targeted region is the intragenic region encompassing the translation
initiation or termination codon of the open reading frame (ORF) of a
gene.

[0089] Another target region includes the 5' untranslated region (5'UTR),
known in the art to refer to the portion of an mRNA in the 5' direction
from the translation initiation codon, and thus including nucleotides
between the 5' cap site and the translation initiation codon of an mRNA
(or corresponding nucleotides on the gene). Still another target region
includes the 3' untranslated region (3'UTR), known in the art to refer to
the portion of an mRNA in the 3' direction from the translation
termination codon, and thus including nucleotides between the translation
termination codon and 3' end of an mRNA (or corresponding nucleotides on
the gene). The 5' cap site of an mRNA comprises an N7-methylated
guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5'
triphosphate linkage. The 5' cap region of an mRNA is considered to
include the 5' cap structure itself as well as the first 50 nucleotides
adjacent to the cap site. Another target region for this invention is the
5' cap region.

[0090] Although some eukaryotic mRNA transcripts are directly translated,
many contain one or more regions, known as "introns." which are excised
from a transcript before it is translated. The remaining (and therefore
translated) regions are known as "exons" and are spliced together to form
a continuous mRNA sequence. In one embodiment, targeting splice sites,
i.e., intron-exon junctions or exon-intron junctions, is particularly
useful in situations where aberrant splicing is implicated in disease, or
where an overproduction of a particular splice product is implicated in
disease. An aberrant fusion junction due to rearrangement or deletion is
another embodiment of a target site. mRNA transcripts produced via the
process of splicing of two (or more) mRNAs from different gene sources
are known as "fusion transcripts", introns can be effectively targeted
using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0091] In an embodiment, the antisense oligonucleotides bind to coding
and/or non-coding regions of a target polynucleotide and modulate the
expression and/or function of the target molecule.

[0092] In an embodiment, the antisense oligonucleotides bind to natural
antisense polynucleotides and modulate the expression and/or function of
the target molecule.

[0093] In an embodiment, the antisense oligonucleotides bind to sense
polynucleotides and modulate the expression and/or function of the target
molecule.

[0094] Alternative RNA transcripts can be produced from the same genomic
region of DNA. These alternative transcripts are generally known as
"variants". More specifically, "pre-mRNA varians" are transcripts
produced from the same genomic DNA that differ from other transcripts
produced from the same genomic DNA in either their start or stop position
and contain both intronic and exonic sequence.

[0095] Upon excision of one or more exon or intron regions, or portions
thereof during splicing, pre-mRNA variants produce smaller "mRNA
variants". Consequently, mRNA variants are processed pre-mRNA variants
and each unique pre-mRNA variant must always produce a unique mRNA
variant as a result of splicing. These mRNA variants are also known as
"alternative splice variants". If no splicing of the pre-mRNA variant
occurs then the pre-mRNA variant is identical to the mRNA variant.

[0096] Variants can be produced through the use of alternative signals to
start or stop transcription. Pre-mRNAs and mRNAs can possess more than
one start codon or stop codon. Variants that originate from a pre-mRNA or
mRNA that use alternative start codons are known as "alternative start
variants" of that pre-mRNA or mRNA. Those transcripts that use an
alternative stop codon are known as "alternative stop variants" of that
pre-mRNA or mRNA. One specific type of alternative stop variant is the
"polyA variant" in which the multiple transcripts produced result from
the alternative selection of one of the "polyA stop signals" by the
transcription machinery, thereby producing transcripts that terminate at
unique polyA sites. Within the context of the invention, the types of
variants described herein are also embodiments of target nucleic acids.

[0097] The locations on the target nucleic acid to which the antisense
compounds hybridize are defined as at least a 5-nucleotide long portion
of a target region to which an active antisense compound is targeted.

[0098] While the specific sequences of certain exemplary target segments
are set forth herein, one of skill in the art will recognize that these
serve to illustrate and describe particular embodiments within the scope
of the present invention. Additional target segments are readily
identifiable by one having ordinary skill in the art in view of this
disclosure.

[0099] Target segments 5-100 nucleotides in length comprising a stretch of
at least five (5) consecutive nucleotides selected from within the
illustrative preferred target segments are considered to be suitable for
targeting as well.

[0100] Target segments can include DNA or RNA sequences that comprise at
least the 5 consecutive nucleotides from the 5'-terminus of one of the
illustrative preferred target segments (the remaining nucleotides being a
consecutive stretch of the same DNA or RNA beginning immediately upstream
of the 5'-terminus of the target segment and continuing until the DNA or
RNA contains about 5 to about 100 nucleotides). Similarly preferred
target segments are represented by DNA or RNA sequences that compose at
least the 5 consecutive nucleotides from the 3'-terminus of one of the
illustrative preferred target segments (the remaining nucleotides being a
consecutive stretch of the same DNA or RNA beginning immediately
downstream of the 3'-terminus of the target segment and continuing until
the DNA or RNA contains about 5 to about 100 nucleotides). One having
skill in the art armed with the target segments illustrated herein will
be able, without undue experimentation, to identify further preferred
target segments.

[0101] Once one or more target regions, segments or sites have been
identified, antisense compounds are chosen which are sufficiently
complementary to the target, i.e., hybridize sufficiently well and with
sufficient specificity, to give the desired effect.

[0102] In embodiments of the invention the oligonucleotides bind to an
antisense strand of a particular target. The oligonucleotides are at
least 5 nucleotides in length and can be synthesized so each
oligonucleotide targets overlapping sequences such that oligonucleotides
are synthesized to cover the entire length of the target polynucleotide.
The targets also include coding as well as non coding regions.

[0103] In one embodiment, it is preferred to target specific nucleic acids
by antisense oligonucleotides. Targeting an antisense compound to a
particular nucleic acid, is a multistep process. The process usually
begins with the identification of a nucleic acid sequence whose function
is to be modulated. This may be, for example, a cellular gene (or mRNA
transcribed from the gene) whose expression is associated with a
particular disorder or disease state, or a non coding polynucleotide such
as for example, non coding RNA (ncRNA).

[0104] RNAs can be classified into (1) messenger RNAs (mRNAs), which are
translated into proteins, and (2) non-protein-coding RNAs (ncRNAs),
ncRNAs comprise microRNAs, antisense transcripts and other
Transcriptional Units (TU) containing a high density of stop codons and
lacking any extensive "Open Reading Frame", Many ncRNAs appear to start
from initiation sites in 3' untranslated regions (3'UTRs) of
protein-coding loci. ncRNAs are often rare and at least half of the
ncRNAs that have been sequenced by the FANTOM consortium seem not to be
polyadenylated. Most researchers have for obvious reasons focused on
polyadenylated mRNAs that are processed and exported to the cytoplasm.
Recently, it was shown that the set of non-polyadenylated nuclear RNAs
may be very large, and that many such transcripts arise from so-called
intergenic regions. The mechanism by which ncRNAs may regulate gene
expression is by base pairings with target transcripts. The RNAs that
function by base pairing can be grouped into (1) cis encoded RNAs that
are encoded at the same genetic location, but on the opposite strand to
the RNAs they act upon and therefore display perfect complementarity to
their target, and (2) trans-encoded RNAs that are encoded at a
chromosomal location distinct from the RNAs they act upon and generally
do not exhibit perfect base-pairing potential with their targets.

[0105] Without wishing to be bound by theory, perturbation of an antisense
polynucleotide by the antisense oligonucleotides described herein can
alter the expression of the corresponding sense messenger RNAs. However,
this regulation can either be discordant (antisense knockdown results in
messenger RNA elevation) or concordant antisense knockdown results in
concomitant messenger RNA reduction). In these cases, antisense
oligonucleotides can be targeted to overlapping or non-overlapping parts
of the antisense transcript resulting in its knockdown or sequestration.
Coding well non-coding antisense can be targeted in an identical manner
and that either category is capable of regulating the corresponding sense
transcripts--either in a concordant or disconcordant manner. The
strategies that are employed in identifying new oligonucleotides for use
against a target can be based on the knockdown of antisense RNA
transcripts by antisense oligonucleotides or any other means of
modulating the desired target.

[0106] Strategy 1: In the case of discordant regulation, knocking down the
antisense transcript elevates the expression of the conventional (sense)
gene. Should that latter gene encode for a known or putative drug target,
then knockdown of its antisense counterpart could conceivably mimic the
action of a receptor agonist or an enzyme stimulant.

[0107] Strategy 2: In the case of concordant regulation, one could
concomitantly knock down both antisense and sense transcripts and thereby
achieve synergistic reduction of the conventional (sense) gene
expression. If, for example, an antisense oligonucleotide is used to
achieve knockdown, then this strategy can be used to apply one antisense
oligonucleotide targeted to the sense transcript and another antisense
oligonucleotide to the corresponding antisense transcript, or a single
energetically symmetric antisense oligonucleotide that simultaneously
targets overlapping sense and antisense transcripts.

[0108] According to the present invention, antisense compounds include
antisense oligonucleotides, ribozymes, external guide sequence (EGS)
oligonucleotides, siRNA compounds, single- or double-stranded RNA
interference (RNAi) compounds such as siRNA compounds, and other
oligomeric compounds which hybridize to at least a portion of the target
nucleic acid and modulate its function. As such, they may be DNA, RNA,
DNA-like, RNA-like; or mixtures thereof, or may be mimetics of one or
more of these. These compounds may be single-stranded, doublestranded,
circular or hairpin oligomeric compounds and may contain structural
elements such as internal or terminal bulges, mismatches or loops.
Antisense compounds are routinely prepared linearly but can be joined or
otherwise prepared to be circular and/or branched. Antiscnse compounds
can include constructs such as, for example, two strands hybridized to
form a wholly or partially double-stranded compound or a single strand
with sufficient self-complementarity to allow for hybridization and
formation of fully or partially double-stranded compound. The two strands
can be linked internally leaving free 3' or 5' termini or can be linked
to form a continuous hairpin structure or loop. The hairpin structure may
contain an overhang on either the 5' or 3' terminus producing an
extension of single stranded character. The double stranded compounds
optionally can include overhangs on the ends. Further modifications can
include conjugate groups attached to one of the termini, selected
nucleotide positions, sugar positions or to one of the internucleoside
linkages. Alternatively, the two strands can be linked via a non-nucleic
acid moiety or linker group. When formed from only one strand, dsRNA can
take the form of a self-complementary hairpin-type molecule that doubles
back on itself to form a duplex. Thus, the dsRNAs can be fully or
partially double stranded. Specific modulation of gene expression can be
achieved by stable expression of dsRNA hairpins in transgenic cell lines,
however, in some embodiments, the gene expression or function is up
regulated. When formed from two strands, or a single strand that takes
the form of a self-complementary hairpin-type molecule doubled back on
itself to form a duplex, the two strands (or duplex-forming regions of a
single strand) are complementary RNA strands that base pair in
Watson-Crick fashion.

[0109] Once introduced to a system the compounds of the invention may
elicit the action of one or more enzymes or structural proteins to effect
cleavage or other modification of the target nucleic acid or may work via
occupancy-based mechanisms. In general, nucleic acids (including
oligonucleotides) may be described as "DNA-like" (i.e., generally having
one or more 2'-deoxy sugars and, generally, T rather than U bases) or
"RNA-like" (i.e., generally having one or more 2'-hydroxyl or 2'-modified
sugars and, generally U rather than T bases). Nucleic acid helices can
adopt more than one type of structure, most commonly the A- and B-forms.
It is believed that, in general, oligonucleotides which have B-form-like
structure are "DNA-like" and those which have A-formlike structure are
"RNA-like." In some (chimeric) embodiments, an antisense compound may
contain both A- and B-form regions.

[0111] dsRNA can also activate gene expression, a mechanism that has been
termed "small RNA-induced gene activation" or RNAa. dsRNAs targeting gene
promoters induce potent transcriptional activation of associated genes.
RNAa was demonstrated in human cells using synthetic dsRNAs, termed
"small activating RNAs" (saRNAs). It is currently not known whether RNAa
is conserved in other organisms.

[0112] Small double-stranded RNA (dsRNA), such as small interfering RNA
(siRNA) and microRNA (miRNA), have been found to be the trigger of an
evolutionary conserved mechanism known as RNA interference (RNAi). RNAi
invariably leads to gene silencing via remodeling chromatin to thereby
suppress transcription, degrading complementary mRNA, or blocking protein
translation. However, in instances described in detail in the examples
section which follows, oligonucleotides are shown to increase the
expression and/or function of the Insulin Receptor Substrate 2 (IRS2)
polynucleotides and encoded products thereof. dsRNAs may also act as
small activating RNAs (saRNA). Without wishing to be bound by theory, by
targeting sequences in gene promoters, saRNAs would induce target gene
expression in a phenomenon referred to as dsRNA-induced transcriptional
activation (RNAa).

[0113] In a further embodiment, the "preferred target segments" identified
herein may be employed in a screen for additional compounds that modulate
the expression of Insulin Receptor Substrate 2 (IRS2) or Transcription
factor E3 (TFE3) polynucleotides. "Modulators" are those compounds that
decrease or increase the expression of a nucleic acid molecule encoding
IRS2 and which comprise at least a 5-nucleotide portion that is
complementary to a preferred target segment. The screening method
comprises the steps of contacting a preferred target segment of a nucleic
acid molecule encoding sense or natural nonsense polynucleotides of IRS2
or TFE3 with one or more candidate modulators, and selecting for one or
more candidate modulators which decrease or increase the expression of a
nucleic acid molecule encoding IRS2 polynucleotides, e.g. SEQ ID NOS: 4
to 9. Once it is shown that the candidate modulator or modulators are
capable of modulating (e.g. either decreasing or increasing) the
expression of a nucleic acid molecule encoding IRS2 polynucleotides, the
modulator may then be employed in further investigative studies of the
function of IRS2 polynucleotides, or for use as a research, diagnostic,
or therapeutic agent in accordance with the present invention.

[0114] Targeting the natural antisense sequence preferably modulates the
function of the target gene. For example, the IRS2 gene (e.g. accession
number NM--003749). In an embodiment, the target is an antisense
polynucleotide of the IRS2 or TFE3 gene. In an embodiment, an antisense
oligonucleotide targets sense and/or natural antisense sequences of IRS2
or TFE3 polynucleotides (e.g. accession number NM--003749) variants,
alleles, isoforms, homologs, mutants, derivatives, fragments and
complementary sequences thereto. Preferably the oligonucleotide is an
antisense molecule and the targets include coding and noncoding regions
of antisense and/or sense IRS2 or TFE3 polynucleotides.

[0115] The preferred target segments of the present invention may be also
be combined with their respective complementary antisense compounds of
the present invention to form stabilized double-stranded (duplexed)
oligonucleotides.

[0116] Such double stranded oligonucleotide moieties have been shown in
the art to modulate target expression and regulate translation as well as
RNA processing via an antisense mechanism. Moreover, the double-stranded
moieties may be subject to chemical modifications. For example, such
double-stranded moieties have been shown to inhibit the target by the
classical hybridization of antisense strand of the duplex to the target,
thereby triggering enzymatic degradation of the target.

[0118] In accordance with embodiments of the invention, the target nucleic
acid molecule is not limited to Insulin Receptor Substrate 2 (IRS2) and
Transcription factor E3 (TFE3) alone but extends to any of the isoforms,
receptors, homologs and the like of IRS2 and TFE3 molecules.

[0119] In an embodiment, an oligonucleotide targets a natural antisense
sequence of IRS2 or TFE3 polynucleotides, for example, polynucleotides
set forth as SEQ ID NOS: 2 and 3, and any variants, alleles, homologs,
mutants, derivatives, fragments and complementary sequences thereto.
Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 4 to
9.

[0120] In one embodiment, the oligonucleotides are complementary to or
bind to nucleic acid sequences of IRS2 or TFE3 antisense, including
without limitation noncoding sense and/or antisense sequences associated
with IRS2 or TFE3 polynucleotides and modulate expression and/or function
of IRS2 molecules.

[0121] In an embodiment, the oligonucleotides are complementary to or bind
to nucleic acid sequences of IRS2 or TFE3 natural antisense, set forth as
SEQ ID NOS: 2 and 3 and modulate expression and/or function of IRS2
molecules.

[0123] The polynucleotide targets comprise IRS2 and TFE3, including family
members thereof, variants of IRS2 and TFE3; mutants of IRS2 and TFE3,
including SNPs; noncoding sequences of IRS2 and TFE3; alleles of IRS2 and
TFE3; species variants, fragments and the like. Preferably the
oligonucleotide is an antisense molecule.

[0125] In an embodiment, targeting of IRS2 or TFE3 polynucleotides, e.g.
SEQ ID NOS: 2 and 3 modulate the expression or function of these targets.
In one embodiment, expression or function is up-regulated as compared to
a control. In an embodiment, expression or function is down-regulated as
compared to a control.

[0126] In an embodiment, antisense compounds comprise sequences set forth
as SEQ ID NOS: 4 to 9. These oligonucleotides can comprise one or more
modified nucleotides, shorter or longer fragments, modified bonds and the
like.

[0127] In an embodiment, SEQ ID NOS: 4 to 9 comprise one or more LNA
nucleotides.

[0128] The modulation of a desired target nucleic acid can be carried out
in several ways known in the art. For example, antisense
oligonucleotides, siRNA etc. Enzymatic nucleic acid molecules (e.g.,
ribozymes) are nucleic acid molecules capable of catalyzing one or more
of a variety of reactions, including the ability to repeatedly cleave
other separate nucleic acid molecules in a nucleotide base
sequence-specific manner. Such enzymatic nucleic acid molecules can be
used, for example, to target virtually any RNA transcript.

[0129] Because of their sequence-specificity, trans-cleaving enzymatic
nucleic acid molecules show promise as therapeutic agents for human
disease (Usman & McSwiggen, (1995) Ann. Rep. Med. Chem. 30, 285-294;
Christoffersen and Marr, (1995) J. Med. Chem. 38, 2023-2037). Enzymatic
nucleic acid molecules can be designed to cleave specific RNA targets
within the background of cellular RNA. Such a cleavage event renders the
mRNA non-functional and abrogates protein expression from that RNA. In
this manner, synthesis of a protein associated with a disease state can
be selectively inhibited.

[0130] In general, enzymatic nucleic acids with RNA cleaving activity act
by first binding to a target RNA. Such binding occurs through the target
binding portion of an enzymatic nucleic acid which is held in close
proximity to an enzymatic portion of the molecule that acts to cleave the
target RNA. Thus, the enzymatic nucleic acid first recognizes and then
binds a target RNA through complementary base pairing, and once bound to
the correct site, acts enzymatically to cut the target RNA. Strategic
cleavage of such a target RNA will destroy its ability to direct
synthesis of an encoded protein. After an enzymatic nucleic acid has
bound and cleaved its RNA target, it is released from that RNA to search
for another target and can repeatedly bind and cleave new targets.

[0131] Several approaches such as in vitro selection (evolution)
strategies (Orgel, (1979) Proc. R. Soc. London, B 205, 435) have been
used to evolve new nucleic acid catalysts capable of catalyzing a variety
of reactions, such as cleavage and ligation of phosphodiester linkages
and amide linkages.

[0132] The development of ribozymes that are optimal for catalytic
activity would contribute significantly to any strategy that employs
RNA-cleaving ribozymes for the purpose of regulating gene expression. The
hammerhead ribozyme, for example, functions with a catalytic rate (kcat)
of about 1 min-1 in the presence of saturating (10 mM) concentrations of
Mg2+ cofactor. An artificial "RNA ligase" ribozyme has been shown to
catalyze the corresponding self-modification reaction with a rate of
about 100 min-1. In addition, it is known that certain modified
hammerhead ribozymes that have substrate binding arms made of DNA
catalyze RNA cleavage with multiple turn-over rates that approach 100
min-1. Finally, replacement of a specific residue within the catalytic
core of the hammerhead with certain nucleotide analogues gives modified
ribozymes that show as much as a 10-fold improvement in catalytic rate.
These findings demonstrate that ribozymes can promote chemical
transformations with catalytic rates that are significantly greater than
those displayed in vitro by most natural self-cleaving ribozymes. It is
then possible that the structures of certain selfcleaving ribozymes may
be optimized to give maximal catalytic activity, or that entirely new RNA
motifs can be made that display significantly faster rates for RNA
phosphodiester cleavage.

[0133] Intermolecular cleavage of an RNA substrate by an RNA catalyst that
fits the "hammerhead" model was first shown in 1987 (Uhlenbeck, O. C.
(1987) Nature, 328: 596-600). The RNA catalyst was recovered and reacted
with multiple RNA molecules, demonstrating that it was truly catalytic.

[0134] Catalytic RNAs designed based on the "hammerhead" motif have been
used to cleave specific target sequences by making appropriate base
changes in the catalytic RNA to maintain necessary base pairing with the
target sequences. This has allowed use of the catalytic RNA to cleave
specific target sequences and indicates that catalytic RNAs designed
according to the "hammerhead" model may possibly cleave specific
substrate RNAs in vivo.

[0135] RNA interference (RNAi) has become a powerful tool for modulating
gene expression in mammals and mammalian cells. This approach requires
the delivery of small interfering RNA (siRNA) either as RNA itself or as
DNA, using an expression plasmid or virus and the coding sequence for
small hairpin RNAs that are processed to siRNAs. This system enables
efficient transport of the pre-siRNAs to the cytoplasm where they are
active and permit the use of regulated and tissue specific promoters for
gene expression.

[0136] In an embodiment, an oligonucleotide or antisense compound
comprises an oligomer or polymer of ribonucleic acid (RNA) and/or
deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog
thereof. This term includes oligonucleotides composed of naturally
occurring nucleotides, sugars and covalent internucleoside (backbone)
linkages as well as oligonucleotides having non-naturally occurring
portions which function similarly. Such modified or substituted
oligonucleotides are often desired over native forms because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for a target nucleic acid and increased stability in the
presence of nucleases.

[0137] According to the present invention, the oligonucleotides or
"antisense compounds" include antisense oligonucleotides (e.g. RNA, DNA,
mimetic, chimera, analog or homolog thereof), ribozymes, external guide
sequence (EGS) oligonucleotides, siRNA compounds, single- or
double-stranded RNA interference (RNAi) compounds such as siRNA
compounds, saRNA, aRNA, and other oligomeric compounds which hybridize to
at least a portion of the target nucleic acid and modulate its function.
As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof,
or may be mimetics of one or more of these. These compounds may be
single-stranded, double-stranded, circular or hairpin oligomeric
compounds and may contain structural elements such as internal or
terminal bulges, mismatches or loops. Antisense compounds are routinely
prepared linearly but can be joined or otherwise prepared to be circular
and/or branched. Antisense compounds can include constructs such as, for
example, two strands hybridized to form a wholly or partially
double-stranded compound or a single strand with sufficient
self-complementarity allow for hybridization and formation of a fully or
partially double-stranded compound. The two strands can be linked
internally leaving free 3' or 5' termini or can be linked to form a
continuous hairpin structure or loop. The hairpin structure may contain
an overhang on either the 5' or 3' terminus producing an extension of
single stranded character. The double stranded compounds optionally can
include overhangs on the ends. Further modifications can include
conjugate groups attached to one of the termini, selected nucleotide
positions, sugar positions or to one of the internucleoside linkages.
Alternatively, the two strands can be linked via a non-nucleic acid
moiety or linker group. When formed from only one strand, dsRNA can take
the form of a self-complementary hairpin-type molecule that doubles back
on itself to form a duplex. Thus, the dsRNAs can be fully or partially
double stranded. Specific modulation of gene expression can be achieved
by stable expression of dsRNA hairpins in transgenic cell lines. When
formed from two sounds, or a single strand that takes the form of a
self-complementary hairpin-type molecule doubled back on itself to form a
duplex, the two strands (or duplex-forming regions of a single strand)
are complementary RNA strands that base pair in Watson-Crick fashion.

[0138] Once introduced to a system, the compounds of the invention may
elicit the action of one or more enzymes or structural proteins to effect
cleavage or other modification of the target nucleic acid or may work via
occupancy-based mechanisms. In general, nucleic acids (including
oligonucleotides) may be described as "DNA-like" (i.e., generally having
one or more 2'-deoxy sugars and, generally, T rather than U bases) or
"RNA-like" (i.e., generally having one or more 2'-hydroxyl or 2'-modified
sugars and, generally U rather than T bases). Nucleic acid helices can
adopt more than one type of structure, most commonly the A- and B-forms.
It is believed that, in general, oligonucleotides which have B-form-like
structure are "DNA-like" and those which have A-formlike structure are
"RNA-like." In some (chimeric) embodiments, an antisense compound may
contain both A- and B-form regions.

[0139] The antisense compounds in accordance with this invention can
comprise an antisense portion from about 5 to about 80 nucleotides (i.e.
from about 5 to about 80 linked nucleosides) in length. This refers to
the length of the antisense strand or portion of the antisense compound.
In other words, a single-stranded antisense compound of the invention
comprises from 5 to about 80 nucleotides, and a double-stranded antisense
compound of the invention (such as a dsRNA, for example) comprises a
sense and an antisense strand or portion of 5 to about 80 nucleotides in
length. One of ordinary skill in the art will appreciate that this
comprehends antisense portions of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 16, 37, 38, 39, 40,41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any
range therewithin.

[0141] In one embodiment, the antisense or oligonucleotide compounds of
the invention have antisense portions of 12 or 13 to 30 nucleotides in
length. One having ordinary skill in the art will appreciate that this
embodies antisense compounds having antisense portions of 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides
in length, or any range therewithin.

[0142] In an embodiment, the oligomeric compounds of the present invention
also include variants in which a different base is present at one or more
of the nucleotide positions in the compound. For example, if the first
nucleotide is an adenosine, variants may be produced which contain
thymidine, guanosine or cytidine at this position. This may be done at
any of the positions of the antisense or dsRNA compounds. These compounds
are then tested using the methods described herein to determine their
ability to inhibit expression of a target nucleic acid.

[0143] In some embodiments, homology, sequence identity or
complementarity, between the antisense compound and target is from about
40% to about 60%. In some embodiments, homology, sequence identity or
complementarity, is from about 60% to about 70%. In some embodiments,
homology, sequence identity or complementarity, is from about 70% to
about 80%. In some embodiments, homolopy, sequence identity or
complementarity, is from about 80% to about 90%. In some embodiments,
homology, sequence identity or complementarity, is about 90%, about 92%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about
100%.

[0144] In an embodiment, the antisense oligonucleotides, such as for
example, nucleic acid molecules set forth in SEQ ID NOS: 2 to 9 comprise
one or more substitutions or modifications. In one embodiment, the
nucleotides are substituted with locked nucleic acids (LNA).

[0145] In an embodiment, the oligonucleotides target one or more regions
of the nucleic acid molecules sense and/or antisense of coding and/or
non-coding sequences associated with IRS2 or TFE3 and the sequences set
forth SEQ ID NOS: 1 to 3. The oligonucleotides are also targeted to
overlapping regions of SEQ ID NOS: 1 to 3.

[0146] Certain preferred oligonucleotides of this invention are chimeric
oligonucleotides. "Chimeric oligonucleotides" or "chimeras," in the
context of this invention, are oligonucleotides which contain two or more
chemically distinct regions, each made up of at least one nucleotide.
These oligenucleotides typically contain at least one region of modified
nucleotides that confers one or more beneficial properties (such as, for
example, increased nuclease resistance, increased uptake into cells,
increased binding affinity for the target) and a region that is a
substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By
way of example, RNase H is a cellular endonuclease which cleaves the RNA
strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the efficiency of
antisense modulation of gene expression. Consequently, comparable results
can often be obtained with shorter oligonucleotides when chimeric
oligonucleotides are used, compared to phosphorothioate
deoxyoligonucleotides hybridizing to the same target region. Cleavage of
the RNA target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known in the
art. In one an embodiment, a chimeric oligonucleotide comprises at least
one region modified to increase target binding affinity, and, usually, a
region that acts as a substrate for RNAse H. Affinity of an
oligonucleotide for its target (in this case, a nucleic acid encoding
ras) is routinely determined by measuring the Tm of an
oligonucleotide/target pair, which is the temperature at which the
oligonucleotide and target dissociate; dissociation is detected
spectrophotometrically. The higher the Tm, the greater is the affinity of
the oligonucleotide for the target.

[0147] Chimeric antisense compounds of the invention may be formed as
composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotides mimetics as
described above. Such; compounds have also been referred to in the art as
hybrids or gapmers. Representative United States patents that teach the
preparation of such hybrid structures comprise, but are not limited to,
U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007, 5,256,775: 5,366,878;
5,403,711; 5,491,133: 5,565,350; 5,623,065; 5,652,355: 5,652,356; and
5,700,922, each of which is herein incorporated by reference.

[0148] In an embodiment, the region of the oligonucleotide which is
modified comprises at least one nucleotide modified at the 2' position of
the sugar, most preferably a 2'-Oalkyl, 2'-O-alkyl-O-alkyl or
2'-fluoro-modified nucleotide. In other an embodiment, RNA modifications
include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose
of pyrimidines, abasic residues or an inverted base at the 3' end of the
RNA. Such modifications are routinely incorporated into oligonucleotides
and these oligonucleotides have been shown to have a higher Tm (i.e.,
higher target binding affinity) than; 2'-deoxyoligonucleotides against a
given target. The effect of such increased affinity is to greatly enhance
RNAi oligonucleotide inhibition of gene expression. RNAse H is a cellular
endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation
of this enzyme therefore results in cleavage of the RNA target, and thus
can greatly enhance the efficiency of RNAi inhibition. Cleavage of the
RNA target can be routinely demonstrated by gel electrophoresis. In an
embodiment, the chimeric oligonucleotide is also modified to enhance
nuclease resistance. Cells contain a variety of exo- and endo-nucleases
which can degrade nucleic acids. A number of nucleotide and nucleoside
modifications have been shown to make the oligonucleotide into which they
are incorporated more resistant to nuclease digestion than the native
oligodeoxynucleotide. Nuclease resistance is routinely measured by
incubating oligonucleotides with cellular extracts or isolated nuclease
solutions and measuring the extent of intact oligonucleotide remaining
over time, usually by gel electrophoresis. Oligonucleotides which have
been modified to enhance their nuclease resistance survive intact for a
longer time than unmodified oligonucleotides. A variety of
oligonucleotide modifications have been demonstrated to enhance or confer
nuclease resistance. Oligonucleotides which contain at least one
phosphorothioate modification are presently more preferred. In some
cases, oligonucleotide modifications which enhance target binding
affinity are also, independently, able to enhance nuclease resistance.

[0149] Specific examples of some preferred oligonucleotides envisioned for
this invention include those comprising modified backbones, for example,
phosphorothioates, phosphotriesters, methyl phosphonates, short chain
alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or
heterocyclic intersugar linkages. Most preferred are oligonucleotides
with phosphorothioate backbones and those with heteroatom backbones,
particularly CH2-NH--O--CH2, CH,--N(CH3)-O--CH2 [known as a
methylene(methylimino) or MMI backbone], CH2-O--N (CH3)-CH2, CH2-N(CH3)-N
(CH3)-CH2 and O--N(CH3)-CH2-CH2 backbones, wherein the native
phosphodiester backbone is represented as O--P--O--CHs). The amide
backbones disclosed by De Mesmacker et al. (1995) Acc. Chem. Res.
28:366-374 are also preferred. Also preferred are oligonucleotides having
morpholino backbone structures (Summerton and Weller, U.S. Pat. No.
5,034,506). In other an embodiment, such as the peptide nucleic acid
(PNA) backbone, the phosphodiester backbone of the oligonucleotide is
replaced with a polyamide backbone, the nucleotides being bound directly
or indirectly to the aza nitrogen atoms of the polyamide backbone.
Oligonucleotides may also comprise one or more substituted sugar
moieties. Preferred oligonucleotides comprise one of the following at the
2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 O(CH2)n CH3, O(CH2)n
NH2 or O(CH2)n CH3 where n is from 1 to about 10; C1 to C10 lower alkyl,
alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN;
CF3; OCF3; O--, S--, or N-alkyl O--, S--, or N-alkenyl; SOCH3; SO2 CH3;
ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl;
aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving
group; a reporter group; an intercalator; a group for improving the
pharmacokinetic properties of an oligonucleotide; or a group for
improving the pharmacodynamic properties of an oligonucleotide and other
substituents having similar properties. A preferred modification includes
2'-methoxyethoxy [2'-O-CH2 CH2 OCH3, also known as
2'-O-(2-methoxyethyl)]. Other preferred modifications include 2'-methoxy
propoxy (2-O--CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on the
oligonucleotide; particularly the 3' position of the sugar on the 3'
terminal nucleotide and the 5' position of 5' terminal nucleotide.
Oligonucleotides may also have sugar mimetics such as cyclobutyls in
place of the pentofuranosyl group.

[0150] Oligonucleotides may also include, additionally or alternatively,
nucleobase (often referred to in the art simply as "base") modifications
or substitutions. As used herein, "unmodified" or "natural" nucleotides
include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil
(U). Modified nucleotides include nucleotides found only infrequently or
transiently in natural nucleic acids, e.g., hypoxanthine,
6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also
referred to as 5-methyl-2' deoxycytosine and often referred to in the art
as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl
HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine,
2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,
2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,
2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,
8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and
2,6-diaminopurine, A "universal" base known in the art, e.g., inosine,
may be included. 5-Me-C substitutions have been shown to increase nucleic
acid duplex stability by 0.6-1.2° C. and are presently preferred
base substitutions.

[0151] Another modification of the oligonucleotides of the invention
involves chemically linking to the oligonucleotide one or more moieties
or conjugates which enhance the activity or cellular uptake of the
oligonucleotide. Such moieties include but are not limited to lipid
moieties such as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or Adamantane acetic acid. Oligonucleotides
comprising Iipophilic moieties, and methods for preparing such
oligonucleotides are known in the art, for example, U.S. Pat. No.
5,138,045, 5,218,105 and 5,459,255.

[0152] It is not necessary for all positions in a given oligonucleotide to
be uniformly modified, and in fact more than one of the aforementioned
modifications may be incorporated in a single oligonucleotide or even at
within a single nucleoside within an oligonucleotide. The present
invention also includes oligonucleotides which are chimeric
oligonucleotides as hereinbefore defined.

[0153] In another embodiment, the nucleic acid molecule of the present
invention is conjugated with another moiety including but not limited to
abasic nucleotides, polyether, polyamine, polyamides, peptides,
carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in the
art will recognize that these molecules can be linked to one or more of
any nucleotides comprising the nucleic acid molecule at several positions
on the sugar, base or phosphate group.

[0154] The oligonucleotides used in accordance with this invention may be
conveniently and routinely made through the well-known technique of solid
phase synthesis. Equipment for such synthesis is sold by several vendors
including Applied Biosystems. Any other means for such synthesis may also
be employed; the actual synthesis of the oligonucleotides is well within
the talents of one of ordinary skill in the art. It is also well known to
use similar techniques to prepare other oligonucleotides such as the
phosphorothioates and alkylated derivatives. It is also well known to use
similar techniques and commercially available modified amidites and
controlled-pore glass (CPG) products such as biotin, fluorescein,
acridine or psoralen-modified amidites and/or CPG (available from Glen
Research, Sterling Va.) to synthesize fluorescently labeled, biotinylated
or other modified oligonucleotides such as cholesterol-modified
oligonucleotides.

[0155] In accordance with the invention, use of modifications such as the
use of LNA monomers to enhance the potency, specificity and duration of
action and broaden the routes of administration of oligonucleotides
comprised of current chemistries such as MOE, ANA, FANA, PS etc. This can
be achieved by substituting some of the monomers in the current
oligonucleotides by LNA monomers. The LNA modified oligonucleotide may
have a size similar to the parent compound or may be larger or preferably
smaller. It is preferred that such LNA-modified oligonucleotides contain
less than about 70%, more preferably less than about 60%, most preferably
less than about 50% LNA monomers and that their sizes are between about 5
and 25 nucleotides, more preferably between about 12 and 20 nucleotides.

[0158] Preferred modified oligonucleotide backbones that do not include a
phosphorus atom therein have backbones that are formed by short chain
alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl
or cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages. These comprise
those having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone
backbones; formacetyI and thioformacetyl backbones; methylene formacetyl
and thioformacetyl backbones; alkene containing backbones; sulfamate
backbones; methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N, O, S
and CH2 component parts.

[0160] In other preferred oligonucleotide mimetics, both the sugar and the
internucleoside linkage, i.e., the backbone, of the nucleotide units are
replaced with novel groups. The base units are maintained for
hybridization with an appropriate nucleic acid target compound. One such
oligomeric compound, an oligonucleotide mimetic that has been shown to
have excellent hybridization properties, is referred to as a peptide
nucleic acid (PNA). In PNA compounds, the sugar-backbone of an
oligonucleotide is replaced with an amide containing backbone, in
particular an aminoethylglycine backbone. The nucleobases are retained
and are bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative United States patents that teach
the preparation of PNA compounds comprise, but are not limited to U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein
incorporated by reference. Further teaching of PNA compounds can be found
in Nielsen, et al. (1991) Science 254, 1497-1500.

[0161] In an embodiment of the invention the oligonucleotides with
phosphorothioate backbones and oligonucleosides with heteroatom
backbones, and in particular
--CH2-NH--O--CH2-,--CH2-N(CH3)-O--CH2-known as a
methylene(methylimino) or MMI backbone,
--CH2-O--N(CH3)-CH2-,--CH2N(CH3)-N(CH3) CH2-and-O--N(CH3)-CH2-CH2-
wherein the native phosphodiester backbone is represented
as-O--P--O--CH2- of the above referenced U.S. Pat. No. 5,489,677, and the
amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone structures of
the above-referenced U.S. Pat. No. 5,034,506.

[0162] Modified oligonucleotides may also contain one or more substituted
sugar moieties. Preferred oligonucleotides comprise one of the following
at the 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl;
O--, S-- or N-alkynyl or O alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C to CO alkyl or C2 to CO
alkenyl and alkynyl. Particularly preferred are O (CH2)n OmCH3,
O(CH2)n,OCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and
O(CH2nON(CH2)nCH3)2 where n and m can be from 1 to about 10. Other
preferred oligonucleotides comprise one of the following at the 2'
position: C to CO, (lower alkyl, substituted lower alkyl, alkaryl,
aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3,
SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and other
substituents having similar properties. A preferred modification
comprises 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) i.e., an alkoxyalkoxy group. A further
preferred modification comprises 2'-dimethylaminooxyethoxy, i.e., a
O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples
herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2-O--CH2-N
(CH2)2.

[0163] Other preferred modifications comprise 2'-methoxy (2'-O CH3),
2'-aminopropoxy (2'-O CH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar
modifications may also be made at other positions on the oligonucleotide,
particularly the 3' position of the sugar on the 3' terminal nucleotide
or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal
nucleotide. Oligonucleotides may also have sugar mimetics such as
cyclobutyl moieties in place of the pentofuranosyl sugar. Representative
United States patents that teach the preparation of such modified sugar
structures comprise, but are not limited to, U.S. Pat. Nos. 4,981,957;
5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446;137; 5,466,786;
5,514,785; 5,519,114; 5,567,811; 5,576,427; 5,591,722; 5,597,909;
5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and
5,700,920, each of which is herein incorporated by reference.

[0164] Oligonucleotides may also comprise nucleobase (often referred to in
the art simply as "base") modifications or substitutions. As used herein,
"unmodified" or "natural" nucleotides comprise the purine bases adenine
(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)
and uracil (U). Modified nucleotides comprise other synthetic and natural
nucleotides such as 5-methylcytosine 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of
adenine and guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-hydroalkyl and other 8-substituted adenines and guanines,
5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted
uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine
and 8-azaadenine, 7-deazaguanine and 7-deazaguanine and 3-deazaguanine
and 3-deazaadenine.

[0166] Representative United States patents that teach the preparation of
the above noted modified nucleotides as well as other modified
nucleotides comprise, but are not limited to U.S. Pat. Nos. 3,687,808, as
well as 4,845,205, 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,507,177; 5,525,711; 5,552,540;
5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which
is herein incorporated by reference.

[0167] Another modification of the oligonucleotides of the invention
involves chemically linking to the oligonucleotide one or more moieties
or conjugates, which enhance the activity, cellular distribution, or
cellular uptake of the oligonucleotide.

[0168] Such moieties comprise but are not limited to, lipid moieties such
as a cholesterol moiety, cholic acid, a thioether, e.g.,
hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,
dodecandiol or undecyl residues, a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a
polyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety,
or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety.

[0170] Drug discovery: The compounds of the present invention can also be
applied in the areas of drug discovery and target validation. The present
invention comprehends the use of the compounds and preferred target
segments identified herein in drug discovery efforts to elucidate
relationships that exist between IRS2 or TFE3 polynucleotides and a
disease state, phenotype, or condition. These methods include detecting
or modulating IRS2 polynucleotides comprising contacting a sample,
tissue, cell, or organism with the compounds of the present invention,
measuring the nucleic acid or protein level of IRS2 polynucleotides
and/or a related phenotypic or chemical endpoint at some time after
treatment, and optionally comparing the measured value to a non-treated
sample or sample treated with a further compound of the invention. These
methods can also be performed in parallel or in combination with other
experiments to determine the function of unknown genes for the process of
target validation or to determine the validity of a particular gene
product as a target for treatment or prevention of a particular disease,
condition, or phenotype.

Assessing Up-Regulation or Inhibition of Gene Expression:

[0171] Transfer of an exogenous nucleic acid into a host cell or organism
can be assessed by directly detecting the presence of the nucleic acid in
the cell or organism. Such detection can be achieved by several methods
well known in the art. For example, the presence of the exogenous nucleic
can be detected by Southern blot or by a polymerase chain reaction (PCR)
technique using primers that specifically amplify nucleotide sequences
associated with the nucleic acid. Expression of the exogenous nucleic
acids can also be measured using conventional methods including gene
expression analysis. For instance, mRNA produced from an exogenous
nucleic acid can be detected and quantified using a Northern blot and
reverse transcription PCR (RT-PCR).

[0172] Expression of RNA from the exogenous nucleic acid can also be
detected by measuring an enzymatic activity or a reporter protein
activity. For example, antisense modulatory activity can be measured
indirectly as a decrease or increase in target nucleic acid expression as
an indication that the exogenous nucleic acid is producing the effector
RNA. Based on sequence conservation, primers can be designed and used to
amplify coding regions of the target genes. Initially, the most highly
expressed coding region from each gene can be used to build a model
control gene, although any coding or non coding region can be used. Each
control gene is assembled by inserting each coding region between a
reporter coding region and its poly(A) signal. These plasmids would
produce an mRNA with a reporter gene in the upstream portion of the gene
and a potential RNAi target in the 3' non-coding region. The
effectiveness of individual antisense oligonucleotides would be assayed
by modulation of the reporter gene. Reporter genes useful in the methods
of the present invention include acetohydroxyacid synthase (AHAS),
alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase
(GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein
(GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),
cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase
(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives
thereof. Multiple selectable markers are available that confer resistance
to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,
kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and
tetracycline. Methods to determine modulation of a reporter gene are well
known in the art, and include, but are not limited to, fluorometric
methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell
Sorting (FACS), fluorescence microscopy), antibiotic resistance
determination.

[0173] IRS2 protein and mRNA expression can be assayed using methods known
to those of skill in the art and described elsewhere herein. For example,
immunoassays such as the ELISA can be used to measure protein levels.
IRS2 ELISA assay kits are available commercially, e.g., from R&D Systems
(Minneapolis, Minn.).

[0174] In embodiments, IRS2 expression (e.g., mRNA or protein) in a sample
(e.g., cells or tissues in vivo or in vitro) treated using an antisense
oligonucleotide of the invention is evaluated by comparison with IRS2
expression in a control sample. For example, expression of the protein or
nucleic acid can be compared using methods known to those of skill in the
art with that in a mock-treated or untreated sample. Alternatively,
comparison with a sample treated with a control antisense oligonucleotide
(e.g., one having an altered or different sequence) can be made depending
on the information desired. In another embodiment, a difference in the
expression or the IRS2 protein or nucleic acid in a treated vs. an
untreated sample can be compared with the difference in expression of a
different nucleic acid (including any standard deemed appropriate by the
researcher, e.g., a housekeeping gene) in a treated sample vs. an
untreated sample.

[0175] Observed differences can be expressed as desired, e.g., in the form
of a ratio or fraction, for use in a comparison with control. In
embodiments, the level of IRS2 mRNA or protein, in a sample treated with
an antisense oligonucleotide of the present invention, is increased or
decreased by about 1.25-fold to about 10-fold or more relative to an
untreated sample or a sample treated with a control nucleic acid. In
embodiments, the level of IRS2 mRNA or protein is increased or decreased
by at least about 1.25-fold, at least about 1.3-fold, at least about
1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least
about 1.7-fold, at least about 1.8-fold, at least about 2-fold, at least
about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least
about 4-fold, at least about 4.5-fold, at least about 5-fold, at least
about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least
about 7-fold, at least about 7.5-fold, at least about 8-fold, at least
about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at
least about 10-fold or more.

Kits, Research Reagents, Diagnostics, and Therapeutics

[0176] The compounds of the present invention can be utilized for
diagnostics, therapeutics, and prophylaxis, and as research reagents and
components of kits. Furthermore, antisense oligonucleotides, which are
able to inhibit gene expression with exquisite specificity, are often
used by those of ordinary skill to elucidate the function of particular
genes or to distinguish between functions of various members of a
biological pathway.

[0177] For use in kits and diagnostics and in various biological systems,
the compounds of the present invention, either alone or in combination
with other compounds or therapeutics, are useful as tools in differential
and/or combinatorial analyses to elucidate expression patterns of a
portion or the entire complement of genes expressed within cells and
tissues.

[0178] As used herein the term "biological system" or "system" is defined
as any organism, cell, cell culture or tissue that expresses, or is made
competent to express products of the IRS2 and TFE3 genes. These include,
but are not limited to, humans, transgenic animals, cells, cell cultures,
tissues, xenografts, transplants and combinations thereof.

[0179] As one non limiting example, expression patterns within cells or
tissues treated with one or more antisense compounds are compared to
control cells or tissues not treated with antisense compounds and the
patterns produced are analyzed for differential levels of gene expression
as they pertain, for example, to disease association, signaling pathway,
cellular localization, expression level, size, structure or function of
the genes examined. These analyses can be performed on stimulated or
unstimulated cells and in the presence or absence of other compounds that
affect expression patterns.

[0181] The compounds of the invention are useful for research and
diagnostics, because these compounds hybridize to nucleic acids encoding
IRS2 or TFE3. For example, oligonucleotides that hybridize with such
efficiency and under such conditions as disclosed herein as to be
effective IRS2 or TFE3 modulators are effective primers or probes under
conditions favoring gene amplification or detection, respectively. These
primers and probes are useful in methods requiring the specific detection
of nucleic acid molecules encoding IRS2 or TFE3 and in the amplification
of said nucleic acid molecules for detection or for use in further
studies of IRS2 or TFE3. Hybridization of the antisense oligonucleotides,
particularly the primers and probes, of the invention with a nucleic acid
encoding IRS2 or TFE3 can be detected by means known in the art. Such
means may include conjugation of an enzyme to the oligonucleotide,
radiolabeling of the oligonucleotide, or any other suitable detection
means. Kits using such detection means for detecting the level of IRS2 or
TFE3 in a sample may also be prepared.

[0182] The specificity and sensitivity of antisense are also harnessed by
those of skill in the art for therapeutic uses. Antisense compounds have
been employed as therapeutic moieties in the treatment of disease states
in animals, including humans. Antisense oligonucleotide drugs have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that antisense
compounds can be useful therapeutic modalities that can be configured to
be useful in treatment regimes for the treatment of cells, tissues and
animals, especially humans.

[0183] For therapeutics, an animal, preferably a human, suspected of
having a disease or disorder which can be treated by modulating the
expression of IRS2 or TFE3 polynucleotides is treated by administering
antisense compounds in accordance with this invention. For example, in
one non-limiting embodiment, the methods comprise the step of
administering to the animal in need of treatment, a therapeutically
effective amount of IRS2 or TFE3 modulator. The IRS2 or TFE3 modulators
of the present invention effectively modulate the activity of the IRS2 or
modulate the expression of the IRS2 protein. In one embodiment, the
activity or expression of IRS2 in an animal is inhibited by about 10% as
compared to a control. Preferably, the activity or expression of IRS2 in
an animal is inhibited by about 30%. More preferably, the activity or
expression of IRS2 in an animal is inhibited by 50% or more. Thus, the
oligomeric compounds modulate expression of IRS2 mRN A by at least 10%,
by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at
least 50%, by at least 60%, by at least 70%, by at least 75%, by at least
80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%,
by at least 99%, or by 100% as compared to a control.

[0184] In one embodiment, the activity or expression of Insulin Receptor
Substrate 2 (IRS2) and/or in an animal is increased by about 10% as
compared to a control. Preferably, the activity or expression of IRS2 in
an animal is increased by about 30%. More preferably, the activity or
expression of IRS2 in an animal is increased by 50% or more. Thus, the
oligomeric compounds modulate expression of IRS2 mRNA by at least 10%, by
at least 50%, by at least 25%, by at least 30%, by a least 40%, by at
least 50%, by at least 60%, by at least 70%, by at least 75%, by at least
80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%,
by at least 99%, or by 100% as compared to a control.

[0185] For example, the reduction of the expression of Insulin Receptor
Substrate 2 (IRS2) may be measured in serum, blood, adipose tissue, liver
or any other body fluid, tissue or organ of the animal. Preferably, the
cells contained within said fluids, tissues or organs being analyzed
contain a nucleic acid molecule encoding IRS2 peptides and/or the IRS2
protein itself.

[0186] The compounds of the invention can be utilized in pharmaceutical
compositions by adding an effective amount of a compound to a suitable
pharmaceutically acceptable diluent or carrier. Use of the compounds and
methods of the invention may also be useful prophylactically.

Conjugates

[0187] Another modification of the oligonucleotides of the invention
involves chemically linking to the oligonucleotide one or more moieties
or conjugates that enhance the activity, cellular distribution or
cellular uptake of the oligomucleotide. These moieties or conjugates can
include conjugate groups covalently bound to functional groups such as
primary or secondary hydroxyl groups. Conjugate groups of the invention
include intercalators, reporter molecules, polyamines, polyamides,
polyethylene glycols, polyethers, groups that enhance the pharmacodynamic
properties of oligomers, and groups that enhance the pharmacokinetic
properties of oligomers. Typical conjugate groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Groups that enhance the pharmacodynamic properties, in the context of
this invention, include groups that improve uptake, enhance resistance to
degradation, and/or strengthen sequence-specific hybridization with the
target nucleic acid. Groups that enhance the pharmacokinetic properties,
in the context of this invention, include groups that improve uptake,
distribution, metabolism or excretion of the compounds of the present
invention. Representative conjugate groups are disclosed in International
Patent Application No. PCT/US92/09196, filed. Oct. 23, 1992, and U.S.
Pat. No. 6,287,860, which we incorporated herein by reference. Conjugate
moieties include, but are not limited to, lipid moieties such as a
cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,
a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl
residues, a phospholipid, e.g., di-hexadecyl-rac-gycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate, a
polyamine or a polyethylene glycol chain, or Adamantane acetic acid, a
palmityl moiety, or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the
invention may also be conjugated to active drug substances, for example,
aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen,
ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a
benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a
barbiturate, cephalosporin, a sulfa drug, an antidiabetic, an
antibacterial or an antibiotic.

[0189] The compounds of the invention may also be admixed, encapsulated,
conjugated or otherwise associated with other molecules, molecule
structures or mixtures of compounds, as for example, liposomes,
receptor-targeted molecules, oral, rectal, topical or other formulations,
for assisting in uptake, distribution and/or absorption. Representative
United States patents that teach the preparation of such uptake,
distribution and/or absorption-assisting formulations include, but are
not limited to U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127;
5,521,291; 5,543,165; 5,547,932, 5,583,020; 5,591,721; 4,426,330;
4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221;
5,356,633; 5,395,619; 5,416,016; 5,417,978, 5,462,854; 5,469,854;
5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and
5,595,756, each of which is herein incorporated by reference.

[0190] Although, the antisense oligonucleotides do not need to be
administered in the context of a vector in order to modulate a target
expression and/or function embodiments of the invention relates to
expression vector constructs for the expression of antisense
oligonucleotides, comprising promoters, hybrid promoter gene sequences
and possess a strong constitutive promoter activity, or a promoter
activity which can be induced in the desired case.

[0191] In an embodiment, invention practice involves administering at
least one of the foregoing antisense oligonucleotides with a suitable
nucleic acid delivery system. In one embodiment, that system includes a
non-viral vector operably linked to the polynucleotide. Examples of such
nonviral vectors include the oligonucleotide alone (e.g. any one or more
of SEQ ID NOS: 4 to 9) or in combination with a suitable protein,
polysaccharide or lipid formulation.

[0193] Additionally preferred vectors include viral vectors, fusion
proteins and chemical conjugates. Retroviral vectors include Moloney
murine leukemia viruses and HIV-based viruses. One preferred HIV-based
viral vector comprises at least two vectors wherein the gag and pol genes
are from an HIV genome and the env acne is from another virus. DNA viral
vectors are preferred. These vectors include pox vectors such as orthopox
or avipox vectors, herpesvirus vectors such as a herpes simplex I virus
(HSV) vector, Adenovirus Vectors and Adeno-associated Virus Vectors.

[0194] The antisense compounds of the invention encompass any
pharmaceutically acceptable salts, esters, or salts of such esters, or
any other compound which, upon administration to an animal, including a
human, is capable of providing (directly or indirectly) the biologically
active metabolite or residue thereof.

[0195] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the compounds of
the invention: i.e., salts that retain the desired biological activity of
the parent compound and do not impart undesired toxicological effects
thereto. For oligonucleotides, preferred examples of pharmaceutically
acceptable salts and their uses are further described, in U.S. Pat. No.
6,287,860, which is incorporated herein by reference.

[0196] The present invention also includes pharmaceutical compositions and
formulations that include the antisense compounds of the invention. The
pharmaceutical compositions of the present invention may be administered
in a number of ways depending upon whether local or systemic treatment is
desired and upon the area to be treated. Administration may be topical
(including ophthalmic and to mucous membranes including vaginal and
rectal delivery), pulmonary, e.g., by inhalation or insufflation of
powders or aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral administration
includes intravenous, intraarterial, subcutaneous, intraperitoneal or
intramuscular injection or infusion; or intracranial, e.g., intrathecal
or intraventricular, administration.

[0197] For treating tissues in the central nervous system, administration
can be made by, e.g., injection or infusion into the cerebrospinal fluid.
Administration of antisense RNA into cerebrospinal fluid is described,
e.g., in U.S. Pat. App. Pub. No. 2007/0117772, "Methods for slowing
familial ALS disease progression," incorporated herein by reference in
its entirety.

[0198] When it is intended that the antisense oligonucleotide of the
present invention be administered to cells in the central nervous system,
administration can be with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier. Injection can be made, e.g., in the enterhinal
cortex or hippocampus. Delivery of neurotrophic factors by administration
of an adenovirus vector to motor neurons in muscle tissue is described
in, e.g., U.S. Pat. No. 6,632,427, "Adenoviral-vector-mediated gene
transfer into medullary motor neurons," incorporated herein by reference.
Delivery of vectors directly to the brain, e.g., the striatum, the
thalamus, the hippocampus, or the substantia nigra, is known in the art
and described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vector, for
the transfer of foreign genes into cells of the central nervous system
particularly in brain," incorporated herein by reference. Administration
can be rapid as by injection or made over a period of time as by slow
infusion or administration of slow release formulations.

[0199] The subject antisense oligonucleotides can also be linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. For example, the antisense oligonucleotide
can be coupled to any substance, known in the art to promote penetration
or transport across the blood-brain barrier, such as an antibody to the
transferrin receptor, and administered by intravenous injection. The
antisense compound can be linked with a viral vector, for example, that
makes the antisense compound more effective and/or increases the
transport of the antisense compound across the blood-brain barrier.
Osmotic blood brain barrier disruption can also be accomplished by, e.g.,
infusion of sugars including, but not limited to, meso erythritol, D(+)
galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-)
fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose,
cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose,
D(+) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-)
fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids
including, but not limited to, glutamine, lysine, arginine, asparagine,
aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine,
methionine, phenylalanine, proline, serine, threonine, tyrosine, valine,
and taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042, "Method for
the delivery of genetic material across the blood brain barrier." U.S.
Pat. No. 6,294,520, "Material for passage through the blood-brain
barrier," and U.S. Pat. No. 6,936,589, "Parenteral delivery systems," all
incorporated herein by reference in their entirety.

[0200] The subject antisense compounds may be admixed, encapsulated,
conjugated or otherwise associated with other molecules, molecule
structures or mixtures of compounds, for example, liposomes,
receptor-targeted molecules, oral, rectal, topical or other formulations,
for assisting in uptake, distribution and/or absorption. For example,
cationic lipids may be included in the formulation to facilitate
oligonucleotide uptake. One such composition shown to facilitate uptake
is LIPOFECTIN (available from GIBCO-BRL, Bethesda, Md.).

[0201] Oligonucleotides with at least one 2'-O-methoxyethyl modification
are believed to be particularly useful for oral administration.
Pharmaceutical compositions and formulations for topical administration
may include transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders. Conventional pharmaceutical
carriers, aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable. Coated condoms, gloves and the like may also be
useful.

[0202] The pharmaceutical formulations of the present invention, which may
conveniently be presented in unit dosage from, may be prepared according
to conventional techniques well known in the pharmaceutical industry.
Such techniques include the step of bringing into association the active
ingredients with the pharmaceutical carrier(s) or excipient(s). In
general, the formulations are prepared by uniformly and intimately
bringing into association the active ingredients with liquid carriers or
finely divided solid carriers or both, and then, if necessary, shaping
the product.

[0203] The compositions of the present invention may be formulated into
any of many possible dosage forms such as, but not hunted to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories, and
enemas. The compositions of the present invention may also be formulated
as suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the viscosity of
the suspension including, for example, sodium carboxymethylcellulose,
sorbitol and/or dextran. The suspension may also contain stabilizers.

[0204] Pharmaceutical compositions of the present invention include, but
are not limited to, solutions, emulsions, foams and liposome-containing
formulations. The pharmaceutical compositions and formulations of the
present invention may comprise one or more penetration enhancers,
carriers, excipients or other active or inactive ingredients.

[0205] Emulsions are typically heterogeneous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1 μm
in diameter. Emulsions may contain additional components in addition to
the dispersed phases and the active drug that may be present as a
solution in either the aqueous phase, oily phase or itself as a separate
phase. Microemulsions are included as an embodiment of the present
invention. Emulsions and their uses are well known in the art and are
further described in U.S. Pat. No. 6,287,860.

[0206] Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome" means
a vesicle composed of amphiphilic lipids arranged in a spherical bilayer
or bilayers. Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous interior
that contains the composition to be delivered. Cationic liposomes are
positively charged liposomes that are believed to interact with
negatively charged DNA molecules to form a stable complex. Liposomes that
are pH-sensitive or negatively-charged are believed to entrap DNA rather
than complex with it. Both cationic and noncationic liposomes have been
used to deliver DNA to cells.

[0207] Liposomes also include "sterically stabilized" liposomes, a term
which, as used herein, refers to liposomes comprising one or more
specialized lipids. When incorporated into liposomes, these specialized
lipids result in liposomes with enhanced circulation lifetimes relative
to liposomeslacking such specialized lipids. Examples of sterically
stabilized liposomes are those in which part of the vesicle-forming lipid
portion of the liposome comprises one or more glycolipids or is
derivatized with one or more hydrophilic polymers, such as a polyethylene
glycol (PEG) moiety. Liposomes and their uses are further described in
U.S. Pat. No. 6,287,860.

[0208] The pharmaceutical formulations and compositions of the present
invention may also include surfactants. The use of surfactants in drug
products, formulations and in emulsions is well known in the art.
Surfactants and their uses are further described in U.S. Pat. No.
6,287,860, which is incorporated herein by reference.

[0209] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic acids,
particularly oligonucleotides. In addition to aiding the diffusion of
non-lipophilic drugs across cell membranes, penetration enhancers also
enhance the permeability of lipophilic drugs. Penetration enhancers may
be classified as belonging to one of five broad categories, i.e.,
surfactants, fatty acids, bile salts, chelating agents, and non-chelating
nonsurfactants. Penetration enhancers and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein by
reference.

[0210] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.

[0212] For topical or other administration, oligonucleotides of the
invention may be encapsulated within liposomes or may form complexes
thereto, in particular to cationic liposomes. Alternatively,
oligonucleotides may be complexed to lipids, in particular to cationic
lipids. Preferred fatty acids and esters, pharmaceutically acceptable
salts thereof, and their uses are further described in U.S. Pat. No.
6,287,860.

[0213] Compositions and formulations for oral administration include
powders or granules, microparticulates, nanoparticulates, suspensions or
solutions in water or non-aqueous media, capsules, gel capsules, sachets,
tablets or minitablets. Thickeners, flavoring agents, diluents,
emulsifiers, dispersing aids or binders may be desirable. Preferred oral
formulations are those in which oligonucleotides of the invention are
administered in conjunction with one or more penetration enhancers
surfactants and chelators. Preferred surfactants include fatty acids
and/or esters or salts thereof, bile acids and/or salts thereof.
Preferred bile acids/salts and fatty acids and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein by
reference. Also preferred are combinations of penetration enhancers, for
example, fatty acids/salts in combination with bile acids/salts. A
particularly preferred combination is the sodium salt of lauric acid,
capric acid and UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the invention may be delivered orally, in granular
form including sprayed dried particles, or complexed to form micro or
nanoparticles. Oligonucleotide complexing agents and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein by reference.

[0214] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous solutions
that may also contain buffers, diluents and other suitable additives such
as, but not limited to, penetration enhancers, carrier compounds and
other pharmaceutically acceptable carriers or excipients.

[0215] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or more
other chemotherapeutic agents that function by a non-antisense mechanism.
Examples of such chemotherapeutic agents include but are not limited to
cancer chemotherapeutic drugs such as daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin,
mafosfamide, ifosfamide, cytosine arabinoside, bischloroethyl-nitrosurea,
busulfan, mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,
hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,
chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclo-phosphoramide, 5-fluororacil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,
vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,
topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol
(DES). When used with the compounds of the invention, such
chemotherapeutic agents may be used individually (e.g., 5-FU and
oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a
period of time followed by MTX and oligonucleotide), or in combination
with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and
oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).
Anti-inflammatory drugs, including but not limited to nonsteroidal
anti-inflammatory drugs and corticosteroids, and antiviral drugs,
including but not limited to ribivirin, vidarabine, acyclovir and
ganciclovir, may also be combined in compositions of the invention.
Combinations of antisense compounds and other non-antisense drugs are
also within the scope of this invention. Two or more combined compounds
may be used together or sequentially.

[0216] In another related embodiment, compositions of the invention may
contain one or more antisense compounds, particularly oligonucleotides,
targeted to a first nucleic acid and one or more additional antisense
compounds targeted to a second nucleic acid target. For example, the
first target may be a particular antiscnse sequence of IRS2 or TFE3, and
the second target may be a region from another nucleotide sequence.
Alternatively, compositions of the invention may contain two or more
antisense compounds targeted to different regions of the same IRS2 or
TFE3 nucleic acid target. Numerous examples of antisense compounds are
illustrated herein and others may be selected from among suitable
compounds known in the art. Two or more combined compounds may be used
together or sequentially.

Dosing:

[0217] The formulation of therapeutic compositions and their subsequent
administration (dosing) is believed to be within the skill of those in
the art. Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting from
several days to several months, or until a cure is effected or a
diminution of the disease state is achieved. Optimal dosing schedules can
be calculated from measurements of drug accumulation in the body of the
patient. Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides, and can
generally be estimated based on EC50s found to be effective in vitro and
in vivo animal models. In general, dosage is from 0.01 μg to 100 g per
kg of body weight, and may be given once or more daily, weekly, monthly
or yearly, or even once every 2 to 20 years. Persons of ordinary skill in
the art can easily estimate repetition rates for dosing based on measured
residence times and concentrations of the drug in bodily fluids or
tissues. Following successful treatment, it may be desirable to have the
patient undergo maintenance therapy to prevent the recurrence of the
disease state, wherein the oligonucleotide is administered in maintenance
doses, ranging from 0.01 μg to 100 g per kg of body weight, once or
more daily, to once every 20 years.

[0218] In embodiments, a patient is treated with a dosage of drug that is
at least about 1, at least about 2, at least about 3, at least about 4,
at least about 5, at least about 6, at least about 7, at least about 8,
at least about 9, at least about 10, at least about 15, at least about
20, at least about 25, at least about 30, at least about 35, at least
about 40, at least about 45, at least about 50, at least about 60, at
least about 70, at least about 80, at least about 90, or at least about
100 mg/kg body weight. Certain injected dosages of antisense
oligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884,
"Antisense modulation of PTP1B expression," incorporated herein by
reference in its entirety.

[0219] While various embodiments of the present invention have been
described above, it should be understood that they have been presented by
way of example only, and not limitation. Numerous changes to the
disclosed embodiments can be made in accordance with the disclosure
herein without departing from the spirit or scope of the invention. Thus,
the breadth and scope of the present invention should not be limited by
any of the above described embodiments.

[0220] All documents mentioned herein are incorporated herein by
reference. All publications and patent documents cited in this
application are incorporated by reference for all purposes to the same
extent as if each individual publication or patent document were so
individually denoted. By their citation of various references in this
document, Applicants do not admit any particular reference is "prior art"
to their invention. Embodiments of inventive compositions and methods are
illustrated in the following examples.

EXAMPLES

[0221] The following non-limiting Examples serve to illustrate selected
embodiments of the invention. It will be appreciated that variations in
proportions and alternatives in elements of the components shown will be
apparent to those skilled in the art and are within the scope of
embodiments of the present invention.

[0222] As indicated above the term "oligonucleotide specific for" or
"oligonucleotide targets" refers to an oligonucleotide having a sequence
(i) capable of forming a stable complex with a portion of the targeted
gene, or (ii) capable of forming a stable duplex with a portion of an
mRNA transcript of the targeted gene.

[0223] Selection of appropriate oligonucleotides is facilitated by using
computer programs that automatically align nucleic acid sequences and
indicate regions of identity or homology. Such programs are used to
compare nucleic acid sequences obtained, for example, by searching
databases such as GenBank or by sequencing PCR products. Comparison of
nucleic acid sequences from a range of species allows the selection of
nucleic acid sequences that display an appropriate degree of identity
between species. In the case of genes that have not been sequenced,
Southern blots are performed to allow a determination of the degree of
identity between genes in target species and other species. By performing
Southern blots at varying degrees of stringency, as is well known in the
art, it is possible to obtain an approximate measure of identity. These
procedures allow the selection of oligonucleotides that exhibit a high
degree of complementarity to target nucleic acid sequences in a subject
to be controlled and a lower degree of complementarity to corresponding
nucleic acid sequences in other species. One skilled in the art will
realize that there is considerable latitude in selecting appropriate
regions of genes for use in the present invention.

[0224] An antisense compound is "specifically hybridizable" when binding
of the compound to the target nucleic acid interferes with the normal
function of the target nucleic acid to cause a modulation of function
and/or activity, and there is a sufficient degree of complementarity to
avoid non-specific binding of the antisense compound to non-target
nucleic acid sequences under conditions in which specific binding is
desired, i.e., under physiological conditions in the case of in vivo
assays or therapeutic treatment, and under conditions in which assays are
performed in the case of in vitro assays

[0225] The hybridization properties of the oligonucleotides described
herein can be determined by one or more in vitro assays as known in the
art. For example, the properties of the oligonucleotides described herein
can be obtained by determination of binding strength between the target
natural antisense and a potential drug molecules using melting curve
assay.

[0226] The binding strength between the target natural antisense and a
potential drug molecule (Molecule) can be estimated using any of the
established methods of measuring the strength of intermolecular
interactions, for example, a melting curve assay.

[0227] Melting curve assay determines the temperature at which a rapid
transition from double-stranded to single-stranded conformation occurs
for the natural antisense/Molecule complex. This temperature is widely
accepted as a reliable measure of the interaction strength between the
two molecules.

[0228] A melting curve assay can be performed using a cDNA copy of the
actual natural antisense RNA molecule or a synthetic DNA or RNA
nucleotide corresponding to the binding site of the Molecule. Multiple
kits containing all necessary reagents to perform this assay are
available (e.g. Applied Biosystems Inc, MeltDoctor kit). These kits
include a suitable buffer solution containing one of the double strand
DNA (dsDNA) binding dyes (such as ABI HRM dyes, SYR Green, SYTO, etc.).
The properties of the dsDNA dyes are such that they emit almost no
fluorescence in free form, but are highly fluorescent when bound to
dsDNA.

[0229] To perform the assay the cDNA or a corresponding oligonucleotide
are mixed with Molecule in concentrations defined by the particular
manufacturer's protocols. The mixture is heated to 95° C. to
dissociate all pre-formed dsDNA complexes, then slowly cooled to room
temperature or other lower temperature defined by the kit manufacturer to
allow the DNA molecules to anneal. The newly formed complexes are then
slowly heated to 95° C. with simultaneous continuous collection of
data on the amount of fluorescence that is produced by the reaction. The
fluorescence intensity is inversely proportional to the amounts of dsDNA
present in the reaction. The data can be collected using a real time PCR
instrument compatible with the kit (e.g.ABI's StepOne Plus Real Time PCR
System or lightTyper instrument, Roche Diagnostics, Lewes, UK).

[0230] Melting peaks are constructed by plotting the negative derivative
of fluorescence with respect to temperature (-d(Fluorescence)/dT) on the
y-axis) against temperature (x-axis) using appropriate software (for
example lightTyper (Roche) or SDS Dissociation Curve, ABI). The data is
analyzed to identify the temperature of the rapid transition from dsDNA
complex to single strand molecules. This temperature is called Tm and is
directly proportional to the strength of interaction between the two
molecules. Typically, Tm will exceed 40° C.

Example 2

Modulation of IRS2 and TFE3 polynucleotides Treatment of HepG2 Cells with
Antisense Oligonucleotides

[0231] HepG2 cells from ATCC (cat#HB-8065) were grown in growth media
(MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV)+10% FBS
(Mediatech cat# MT35-011-CV)+ penicillin/streptomycin (Mediatech
cat#MT30-002-CI)) at 37° C. and 5%, CO2. One day before the
experiment the cells were replaced at the density of 1.5×105/ml
into 6 well plates and incubated at 37° C. and 5% CO2. On the day
of the experiment the media in the 6 well plates was changed to fresh
growth media. All antisense oligonucleotides were diluted to the
concentration of 20 μM. Two μl of this solution was incubated with
400 μl of Opti-MEM media (Gibco cat#31985-070) and 4 μl of
Lipofectamine 2000 (Invitrogen cat#11668019) at room temperature for 20
min and applied to each well of the 6 well plates with HepG2 cells.
Similar mixture including 2 μl of water instead of the oligonucleotide
solution was used for the mock-transfected controls. After 3-18 h of
incubation at 37° C. and 5% CO2 the media was changed to fresh
growth media. 48 h after addition of antisense oligonucleotides the media
was removed and RNA was extracted from the cells using SV Total RNA
Isolation System from Promega (cat #Z3105) or RNeasy Total RNA Isolation
kit from Qiagen (cat#74181) following the manufacturers' instructions.
600 ng of RNA was added to the reverse transcription reaction performed
using Verso cDNA kit from Thermo Scientific (cat#AB1453B) or High
Capacity cDNA Reverse Transcription Kit (cat#4368813) as described in the
manufacturer's protocol. The cDNA from this reverse transcription
reaction was used to monitor gene expression by real time PCR using ABI
Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by
ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00275843_s1
(IRS2) and Hs00232406_m1 (TFE3) by Applied Biosystems Inc., Foster City
Calif.). The following PCR cycle was used: 50° C. for 2 mm,
95° C. for 10 min, 40 cycles of (95° C. for 15 seconds,
60° C. for 1 min) using StepOne Plus Real Time PCR Machine
(Applied Biosystems). Fold change in gene expression after treatment with
antisense oligonucleotides was calculated based on the difference in
18S-normalized dCt values between treated and mock-transfected samples.

Results:

[0232] Real Time PCR results show that levels of IRS2 mRNA in HepG2 cells
are significantly increased 48 h after treatment with siRNAs to TFE3
antisense Hs.708291 (FIG. 1).

[0233] Real Time PCR results show the fold change+standard deviation in
TFE3 mRNA after treatment of HepG2 cells with siRNA oligonucleotides
introduced using Lipofectamine 2000, as compared to control (FIG. 4).

Treatment of 518A2 Cells with Antisense Oligonucleotides:

[0234] 518A2 cells obtained from Albert Einstein-Montefiore Cancer Center,
NY were grown in growth media (MEM/EBSS (Hyclone cat #SH30024, Mediatech
cat #MT-10-010-CV)+10% FBS (Mediatech
cat#MT35-011-CV)+penicillin/streptomycin (Mediatech cat#MT30-002-CI)) at
37° C. and 5% CO2. One day before the experiment the cells were
replated at the density of 1.5×105/ml into 6 well plates and
incubated at 37° C. and 5% CO2. On the day of the experiment the
media in the 6 well plates was changed to fresh growth media. All
antisense oligonucleotides were diluted to the concentration of 20 μM.
Two μl of this solution was incubated with 400 μl of Opti-MEM media
(Gibco cat#31985-070) and 4 μl of Lipefectamine 2000 (Invitrogen
cat#11668019) at room temperature for 20 min and applied to each well of
the 6 well plates with 518A2 cells. Similar mixture including 2 μl of
water instead of the oligonucleotide solution was used for the
mock-transfected controls. After 3-18 h of incubation at 37° C.
and 5%, CO2 the media was changed to fresh growth media, 48 h after
addition of antisense oligonucleotides the media was removed and RNA was
extracted from the cells using SV Total RNA Isolation System from Promega
(cat #Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181)
following the manufacturers' instructions. 600 ng of RNA was added to the
reverse transcription reaction performed using Verso cDNA kit from Thermo
Scientific (cat#AB1453B) or High Capacity cDNA Reverse Transcription Kit
(cat#4368813 as described in the manufacturer's protocol. The cDNA from
this reverse transcription reaction was used to monitor gene expression
by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and
primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression
Assay: Hs00275843_s1 (IRS2) and Hs00232406_m1 (TFE3) by Applied
Biosystems Inc., Foster City Calif.). The following PCR cycle was used:
50° C. for 2 min, 95° C. for 10 min, 40 cycles of
(95° C. for 15 seconds, 60° C. for 1 min) using StepOne
Plus Real Time PCR Machine (Applied Biosystems). Fold change in gene
expression after treatment with antisense oligonucleotides was calculated
based on the difference in 18S-normalized dCt values between treated and
mock-transfected samples.

[0235] Results: Real Time PCR results show that levels of IRS2 mRNA in
518A2 cells are significantly increased 48 h after treatment with siRNAs
to TFE3 antisense Hs.708291 (FIG. 1).

Treatment Vero76 Cells with Antisense Oligonucleotides:

[0236] Vero76 cells from ATCC (cat#CRL-1587) were grown in growth media
(MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV)+10% FBS
(Mediated cat#MT35-011-CV)+penicillin/streptomycin (Mediatech
cat#MT30-002-CI)) at 37° C. and 5% CO2. One day before the
experiment the cells were replated at the density of
1.5×105/ml into 6 well plates and incubated at 37° C.
and 5% CO2. On the day of the experiment the media in the 6 well plates
was changed to fresh growth media. All antisense oligonucleotides were
diluted in water to the concentration of 20 μM. 2 μl of this
solution was incubated with 400 μl of Opti-MEM media (Gibco
cat#31985-070) and 4 ul of Lipofectamine 2000 (Invitrogen cat#11668019)
at room temperature for 20 min and applied to each well of the 6 well
plates with Vero76 cells. Similar mixture including 2 μl of water
instead of the oligonucleotide solution was used for the mock-transfected
controls. After 3-18 h of incubation at 37° C. and 5% CO2 the
media was changed to flesh growth media, 48 h after addition of antisense
oligonucleotides the media was removed and RNA was extracted from the
cells using SV Total RNA Isolation System from Promega (cat #Z3105) or
RNeasy Total RNA Isolation kit from Qiagen (cat#74181), following the
manufacturers' instructions. 600 ng of RNA was added to the reverse
transcription reaction performed using Verso cDNA kit from Thermo
Scientific (cat#AB1453B) as described in the manufacturer's protocol. The
cDNA from this reverse transcription reaction was used to monitor gene
expression by real time PCR using ABI Taqman Gene Expression Mix
(cat#4369510) and primers/probes designed by ABI (Applied Biosystems
Taqman Gene Expression Assay: Hs00275843_s1 (IRS2) and Hs00232406_m1
(TFE3) by Applied Biosystems Inc., Foster City Calif.). The following PCR
cycle was used: 50° C. for 2 min, 95° C. for 10 min, 40
cycles of (95° C. for 15 seconds, 60° C. for 1 min) using
StepOne Plus Real Time PCR Machine (Applied Biosystems). Fold change in
gene expression after treatment with antisense oligonucleotides was
calculated based on the difference in 18S-normalized dCt values between
treated and mock-transfected samples.

[0237] Results: Real Time PCR results show the fold change+standard
deviation in IRS2 mRNA after treatment of Vero76 cells with
phosphorothioate oligonucleotides introduced using Lipofectamine 2000, as
compared to control (FIG. 2).

Treatment of MCF-7 Cells with Antisense Oligonucleotides:

[0238] MCF-7 cells from ATC (cat#HTB-22) were grown in growth media
(MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV)+10% FBS
(Mediatech cat#MT35-011-CV)+penicillin/streptomycin (Mediatech
cat#MT30-002-CI)) at 37° C. and 5% CO2. One day before the
experiment the cells were replated at the density of 1.5×105/ml
into 6 well plates and incubated at 37° C. and 5% CO2. On the day
of the experiment the media in the 6 well plates was changed to fresh
growth media. All antisense oligonucleotides were diluted to the
concentration of 20 μM. Two μl of this solution was incubated with
400 μl of Opti-MEM media (Gibco cat#31985-070) and 4 μl of
Lipofectamine 2000 (Invitrogen cat#11668019) at room temperature for 20
min and applied to each well of the 6 well plates with MCF-7 cells.
Similar mixture including 2 μl of water instead of the oligonucleotide
solution was used for the mock-transfected controls. After 3-18 h of
incubation at 37° C. and 5% CO2 the media was changed to fresh
growth media. 48 h after addition of antisense oligonucleotides the media
was removed and RNA was extracted from the cells using SV Total RNA
Isolation System from Promega (cat #Z3105) or RNeasy Total RNA Isolation
kit from Qiagen (cat#74181) following the manufacturers' instructions.
600 ng of RNA was added to the reverse transcription reaction performed
using Verso cDNA kit from Thermo Scientific (cat#AB1453B) or High
Capacity cDNA Reverse Transcription Kit (cat#4368813) as described in the
manufacturer's protocol. The cDNA from this reverse transcription
reaction was used to monitor gene expression by real time PCR using ABI
Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by
ABI (Applied Biosystems Taqman Gene Expression Assay: Hs002.75843_s1
(IRS2) and Hs00232406_m1 (TFE3) by Applied Biosystems Inc., Foster City
Calif.). The following PCR cycle was used: 50° C. for 2 min,
95° C. for 10 min, 40 cycles of (95° C. for 15 seconds,
60° C. for 1 min) using StepOne Plus Real Time PCR Machine
(Applied Biosystems). Fold change in gene expression after treatment with
antisense oligonucleotides was calculated based on the difference in
18S-normalized dCt values between treated and mock-transfected samples.

[0239] Results: Real Time PCR results show the fold change+standard
deviation in IRS2 mRNA after treatment of MCF7 cells with
phosphorothioate oligonucleotides introduced using Lipofectamine 2000, as
compared to control (FIG. 3).

[0240] Although the invention has been illustrated and described with
respect to one or more implementations, equivalent alterations and
modifications will occur to others skilled in the art upon the reading
and understanding of this specification and the annexed drawings. In
addition, while a particular feature of the invention may have been
disclosed with respect to only one of several implementations, such
feature may be combined with one or more other features of the other
implementations as may be desired and advantageous for any given or
particular application.

[0241] The Abstract of the disclosure will allow the reader to quickly
ascertain the nature of the technical disclosure. It is submitted with
the understanding that it will not be used to interpret or limit the
scope or meaning of the following claims.